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At least 19 recordsLinked to original sources
PubMed2006

A reducing and denaturing step maximizes the immunoprecipitations of m-calpain and I-2(PP2A)/SET: an approach toward antibodies that do not work well in immunoprecipitation.

Immunoprecipitation is an elegant method to isolate a specific protein of interest from a complex protein mixture such as cell lysate. We tried to increase the efficiency of m-calpain immunoprecipitation with anti-m-calpain antibodies directed toward denatured antigens that only work for immunoblotting and immunohistochemistry. We found that a reducing and denaturing step prior to immunoprecipitation greatly potentiates the efficiency of the immunoreaction. This improved method is also applicable for the immunoprecipitation of oncoprotein I-2(PP2A)/SET with antibodies directed toward a synthetic peptide that only work for immunoblotting. Thus, our improved method provides a way to maximize immunoprecipitation when using antibodies that do not work well under conventional immunoprecipitation conditions. Furthermore, the improved method is also suitable for decreasing the contaminating proteins during immunoprecipitation.

Antibodies↗
PubMed2006

Assessment of multiple different estrogen receptor-beta antibodies for their ability to immunoprecipitate under chromatin immunoprecipitation conditions.

Several different antibodies to total estrogen receptor (ER)beta, ERbeta1 and ERbeta2/cx have been tested and compared for their ability to immunoprecipitate ERbeta specific isoforms under chromatin immunoprecipitation conditions (ChIP). The rabbit polyclonal antibodies AP-ERbeta1 and AP-ERbeta2/cx, specific for ERbeta1 and ERbeta2/cx isoforms, respectively, were the most efficient for ChIP. The monoclonal antibody MCA1974/PPG5/10 was also able to ChIP ERbeta1, but less efficiently than AP-ERbeta1. All other antibodies tested were not suitable for ChIP analyses although most antibodies tested immunoprecipitated the appropriate ERbeta isoforms under standard conditions. To identify antibodies that can also be used to verify in-vivo expression profiles, a comparison of the antibodies to detect ERbeta isoforms by western blotting and immunohistochemistry was also undertaken. Under the tissue processing and autostaining conditions used at the Manitoba Breast Tumor Bank 385P/GC17, MCA1974/PPG5/10, Ab288/14C8 and MCA2279S/57/3 were found to be the best for IHC of ERbeta isoforms in human breast tissue biopsy sections, while Ab14021, AP-ERbeta1 and AP-ERbeta2/cx were best for western blot detection of ERbeta isoforms.

Animals↗
PubMed1993

Identification of p125, a component of a group of 120-kDa proteins that are phosphorylated on tyrosine residues in response to bradykinin and bombesin stimulation, in anti-ras-GTPase-activating protein immunoprecipitates of Swiss 3T3 cells.

Bradykinin (BK) and bombesin (BN) stimulate an increase in the tyrosine phosphorylation of a 120-kDa group of proteins (pps120) in Swiss 3T3 cells (Leeb-Lundberg, L. M. F., and Song X.-H. (1991) J. Biol. Chem. 266, 7746-7749). Here, we show that a component of pps120, p125, was specifically immunoprecipitated with antibodies against the p21ras GTPase-activating protein (GAP). The major portion of GAP in nonstimulated cells (96%) was located in the cytosol, and this distribution was not affected by exposure of cells to 1 microM BK for 1 min. A significant amount of GAP in nonstimulated cells was recovered in anti-phosphotyrosine (anti-Tyr(P)) immunoprecipitates, and the cellular distribution of this GAP essentially paralleled that of total GAP. Recovery of GAP in anti-Tyr(P) immunoprecipitates of nonstimulated cells appeared to be caused at least in part by the presence of GAP complexed to a 190-kDa tyrosine-phosphorylated protein (p190). Exposure of cells to 1 microM BK for 1 min resulted in an increase in the recovery of a portion of the cellular GAP in anti-Tyr(P) immunoprecipitates. This increase was paralleled by the appearance of a tyrosine-phosphorylated protein species of 125 kDa (p125) in anti-GAP immunoprecipitates. Tyrosine-phosphorylated p125 was present also in anti-GAP immunoprecipitates after exposure of cells to 1 microM BN. High performance gel exclusion liquid chromatography of the anti-GAP-immunoprecipitated proteins on a Protein-Pak 300SW column revealed that p125 is not GAP. Anti-GAP immunoprecipitation of p125 was prevented by prior denaturation of cell lysates in sodium dodecyl sulfate suggesting that p125 is physically associated with GAP. Chromatography of cell lysates revealed that the pps120 group of tyrosine phosphoproteins includes a 125- and a 120-kDa protein. The anti-GAP-immunoprecipitable p125 migrated identically to the 125-kDa phosphoprotein component of pps120. These observations show that the pps120 group of tyrosine phosphoproteins is composed of at least two physically distinct protein components, p125 and p120. p125 is associated in some manner with a portion of the cellular GAP after exposure of cells to BK and BN.

3T3 Cells↗
PubMed1994

Pr-specific phytochrome phosphorylation in vitro by a protein kinase present in anti-phytochrome maize immunoprecipitates.

Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.

Antibodies↗
PubMed1989

Determination of changes in the phosphorylation state of the neuron-specific protein kinase C substrate B-50 (GAP43) by quantitative immunoprecipitation.

To determine changes in the degree of phosphorylation of the protein kinase C substrate B-50 in vivo, a quantitative immunoprecipitation assay for B-50 (GAP43, F1, pp46) was developed. B-50 was phosphorylated in intact hippocampal slices with 32Pi or in synaptosomal plasma membranes with [gamma-32P]ATP. Phosphorylated B-50 was immunoprecipitated from slice homogenates or synaptosomal plasma membranes using polyclonal anti-B-50 antiserum. Proteins in the immunoprecipitate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the incorporation of 32P into B-50 was quantified by densitometric scanning of the autoradiogram. Only a single 48-kilodalton phosphoband was detectable in the immunoprecipitate, but this band was absent when preimmune serum was used. The B-50 immunoprecipitation assay was quantitative under the following condition chosen, as (1) recovery of purified 32P-labelled B-50 added to slice homogenates or synaptosomal plasma membranes was greater than 95%; and (2) modulation of B-50 phosphorylation in synaptosomal plasma membranes with adrenocorticotrophic hormone, polymyxin B, or purified protein kinase C in the presence of phorbol diester resulted in EC50 values identical to those obtained without immunoprecipitation. With this immunoprecipitation assay we found that treatment of hippocampal slices with 4 beta-phorbol 12,13-dibutyrate stimulated B-50 phosphorylation, whereas 4 alpha-phorbol 12,13-didecanoate was inactive. Thus, we conclude that the B-50 immunoprecipitation assay is suitable to monitor changes in B-50 phosphorylation in intact neuronal tissue.

Animals↗
PubMed2002

Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo.

Protein-RNA interactions play indispensable structural, catalytic, and regulatory roles within the cell. Understanding their physical association in vivo provides valuable insight into their assembly, function, and regulation in the cellular milieu. Inspired by the chromatin immunoprecipitation assay, we have developed a ribonucleoprotein (RNP) immunoprecipitation assay to study RNA-protein interactions in vivo. This method takes advantage of the highly reactive, reversible crosslinker formaldehyde, combined with high-stringency immunoprecipitation to identify specific RNAs associated with a given protein. The RNP immunoprecipitation (RIP) assay was developed using RNA-protein interactions of hepatitis delta virus (HDV) as a model system. HDV is an RNA virus with a single-stranded circular RNA genome that encodes one viral protein, hepatitis delta antigen (HDAg). The high affinity of HDAg for the HDV RNA genome, combined with the well-characterized anti-HDAg antibodies, made this system a logical starting point for the development of the RIP assay. Cells with replicating HDV were crosslinked with formaldehyde and the HDV RNPs were immunoprecipitated using anti-HDAg antibodies. The crosslinks were then reversed by heat treatment, and the immunoprecipitated HDV RNAs were identified by reverse transcription polymerase chain reaction (RT-PCR). The specificity of this assay was tested using HDV mutants and heterologous antibodies for immunoprecipiation followed by RT-PCR with HDV-specific primers. This experiment showed no nonspecific immunoprecipitation of the HDV RNPs. The method was tested further using protein-RNA interactions known to exist in the U1 snRNP. The results indicate that the RIP assay is a powerful tool to identify RNA-protein interactions in vivo and has the potential to unravel the cellular network of RNP complexes in their native setting.

Cross-Linking Reagents↗
PubMed1993

Eubacterial components similar to small nuclear ribonucleoproteins: identification of immunoprecipitable proteins and capped RNAs in a cyanobacterium and a gram-positive eubacterium.

Small nuclear ribonucleoprotein (snRNP) particles play an important role in the processing of pre-mRNA. snRNPs have been identified immunologically in a variety of cells, but none have ever been observed in prokaryotic systems. This report provides the first evidence for the presence of snRNP-like components in two types of prokaryotic cells: those of the cyanobacterium Synechococcus leopoliensis and those of the gram-positive eubacterium Bacillus subtilis. These components consist of snRNP-immunoreactive proteins and RNAs, including some with the snRNP-unique 5' m2,2,7G (m3G) cap. Immunoreactivity was determined by immunoprecipitation procedures, with either antinuclear-antibody-positive (RNP- and Sm-monospecific) patient sera or a m3G monoclonal antibody, with radiolabelled cell extracts that were preadsorbed with antinuclear-antibody-negative sera. S. leopoliensis immunoprecipitates showed the presence of high-molecular-mass proteins (14 to 70 kDa) and RNAs (138 to 243 nucleotides) that are analogous in size to proteins and RNAs found in human (HEp-2) cell immunoprecipitates but absent in Escherichia coli immunoprecipitates. Thin-layer chromatography of S. leopoliensis immunoprecipitates confirmed the presence of a capped nucleotide similar to a capped nucleotide in HEp-2 immunoprecipitates; no such nucleotide was observed in E. coli immunoprecipitates. Immunoreactive RNAs (117-170 nucleotides) were identified in a second eubacterium, B. subtilis, as well. This work suggests that snRNPs or their evolutionary predecessors predate the emergence of eukaryotic cells.

Antibodies, Antinuclear↗
PubMed1984

Immunoprecipitation of radiolabeled human thyroid cell proteins by serum from patients with autoimmune thyroid disease.

Characterization of the antigen to thyroid-stimulating immunoglobulin (TSI), presumably the TSH receptor, remains elusive. We, therefore, attempted immunoprecipitation of the TSI antigen using cultured human thyroid cells radiolabeled to high specific activity by [35S]methionine (all proteins labeled) or by 125I and lactoperoxidase (primarily cell surface proteins labeled). Both techniques produced similar results. Crude membrane preparations from the labeled cells were solubilized in buffer containing 1% Triton and then incubated with TSI-containing or control serum. Immunoglobulin was precipitated with goat antihuman immunoglobulin and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. About 30 proteins were immunoprecipitated by both normal and control sera. However, only 1 protein (mol wt, approximately 320,000) was preferentially selected by TSI-containing serum (6 of 6 tested). This protein was also immunoprecipitated by the majority of sera from patients with Hashimoto's thyroiditis. The 320,000 mol wt protein constituted only a minute amount of total radiolabeled membrane protein, because it was not apparent on electrophoresis of the membrane extract before immunoprecipitation. It corresponded (in migration) to a human thyroglobulin (Tg) standard. Tg also inhibited immunoprecipitation of the 320,000 mol wt band. The anti-Tg antibody titer correlated significantly with the degree of immunoprecipitation of the 320,000 mol wt band. No specific immunoprecipitation of any protein was observed when radiolabeled guinea pig membrane proteins were used. These data provide insight into the difficulties involved in the identification of the TSH receptor with serum containing TSI.

Antigens↗
PubMed1992

Immunoprecipitation of alpha 2a-adrenergic receptor-GTP-binding protein complexes using GTP-binding protein selective antisera. Changes in receptor/GTP-binding protein interaction following agonist binding.

The nature of the interaction of cloned alpha 2a-adrenergic receptors from LLC-PK1-O clone cells with G proteins was investigated using an immunoprecipitation approach. Following solubilization of the alpha 2a receptors, antiserum 8730, which is directed against the C-terminal region of Gi alpha, immunoprecipitated alpha 2a receptor-Gi alpha complexes. The immunoprecipitation was specific since it could be blocked by the peptide to which antiserum 8730 was generated. Antisera 3646 (anti-Gi alpha 1), 1521 (anti-Gi alpha 2), and 1518 (anti-Gi alpha 3) immunoprecipitated solubilized alpha 2a receptor-Gi alpha complexes, indicating that all three Gi alpha subtypes couple with the alpha 2a receptor. Antiserum 9072, which is directed against the C-terminal region of G(o)alpha, immunoprecipitated solubilized alpha 2a receptor-G alpha complexes indicating that these receptors are also coupled to G(o)alpha. Antiserum 8132, which is directed against G beta 36, immunoprecipitated solubilized alpha 2a receptors while the G beta 35 antiserum 8129, did not, indicating that alpha 2a receptors selectively associate with G beta 36. The binding of the partial agonist p-aminoclonidine to the solubilized alpha 2a receptor alters the association of the receptor with G proteins. Following p-aminoclonidine binding to the solubilized alpha 2a receptor, the ability of the C-terminal directed G alpha antisera 8730 and 9072 to coimmunoprecipitate the alpha 2a receptor-G alpha complex was greatly reduced. The effect of p-aminoclonidine was concentration dependent, mimicked by the full agonist UK 14304 and blocked by the alpha 2 receptor antagonist yohimbine. In contrast, antisera directed against internal regions of Gi alpha and G(o)alpha, immunoprecipitated the agonist-bound and agonist-free alpha 2a receptor equally well. These findings indicate that following the binding of agonists to the alpha 2a receptor, Gi alpha and G(o)alpha remain physically associated with the receptor but either the conformation of G alpha linked to the receptor or the conformation of the receptor itself is modified such that the epitope for the C-terminal directed anti-Gi alpha and anti-G(o)alpha antisera are not accessible. These agonist-induced conformational changes in the alpha 2a receptor-G alpha complex may be important for the activation of the G protein and the stimulation of the alpha 2a receptor signal transduction pathway.

Adrenergic alpha-Agonists↗
PubMed2005

[Screening tumor antigens of ovarian carcinoma by Western blot, immunoprecipitation, and mass spectrometry].

BACKGROUND & OBJECTIVE: Screening tumor antigens plays a key role in early diagnosis of ovarian carcinoma. Several technologies, such as phage antibody library, ribosome display, and serological analysis of recombinant cDNA expression libraries (SEREX), have been applied widely. This study was to screen and identify tumor antigens of ovarian carcinoma by Western blot, immunoprecipitation, and mass spectrometry, and to establish a new methodological entity. METHODS: Western blot was used to find out the obviously different serum in ovarian carcinoma patients; SKOV3 cells lysate was added in the serum; immunoprecipitation was used to enrich antigens; then, the different protein band was gained when compared with normal serum immunoprecipitates, identified by mass spectrometry, and analyzed with bioinformatics. RESULTS: The serum of patient No. 7 was the obviously different one screened with Western blot. Immunoprecipitation and SDS-PAGE showed a different protein band in the serum appeared in the position of about 66.2 ku when compared with normal serum immunoprecipitates. Through mass spectrometry and bioinformatics analysis, heat shock protein 70 (HSP70) and cytokeratin 9 were identified in the different protein band. CONCLUSION: Western blot, immunoprecipitation, and mass spectrometry is an effectively methodological entity for screening and identifying tumor antigens.

Antigens, Neoplasm↗
PubMed1991

The use of rabbit polyclonal antibodies for the isolation of carcinogen-adducted DNA by immunoprecipitation.

Polyclonal rabbit antibodies elicited against DNA with high levels of (+/-) 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) adducts were used to isolate DNA fragments modified by this carcinogen. DNA treated in vitro with different concentrations of BPDE-I was used as substrate in double-antibody immunoprecipitation reactions. The IgG fraction from immune rabbit serum (primary antibody) was reacted with single-stranded plasmid DNA bearing BPDE-I adducts, and the complexes were immunoprecipitated using goat antirabbit-IgG as secondary antibody. DNA was isolated from the immunoprecipitated pellet, blotted onto nitrocellulose or nylon, and hybridized with 32P-labeled sequences homologous to a fragment of the plasmid DNA used in the assay. The recovery of both DNA and adducts in the immunoprecipitated pellet increased with the level of carcinogen adduction of the DNA. The immunoprecipitation reaction appeared to be more efficient for fragments of DNA containing a high number of adducts. The amount of 32P-hybridizing material recovered by immunoprecipitation was virtually identical to the amount added to the reaction in DNA samples that contained three adducts per 10(3) nucleotides.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Increased tyrosine kinase activity in pp60c-src immunoprecipitate from platelet activating factor stimulated human platelets: in vitro phosphorylation of a synthetic peptide.

The involvement of pp60c-src tyrosine kinase was studied in human platelets stimulated with platelet activating factor (PAF). Immunoprecipitation of pp60c-src from platelets followed by immunoblot with pp60v-src monoclonal antibody revealed four protein bands of 60, 56, 50 and 29 kDa as detected by enzymographic web. The phosphorylation of these bands was increased in the pp60c-src immunoprecipitate from PAF stimulated platelets. To assay the tyrosine kinase activity, we used a 13 amino acid synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg- Gly) which contains sequences similar to the phosphorylation site on pp60c-src. Incubation of the pp60c-src immunoprecipitate with the peptide and [32P]ATP caused phosphorylation of this peptide in vitro. This peptide phosphorylation was not observed when normal mouse IgG-bound protein(s) was used instead of pp60c-src immunoprecipitate. The peptide phosphorylation was markedly increased by pp60c-src immunoprecipitate obtained from PAF treated platelets. Lyso-PAF had no effect on the phosphorylation. PAF antagonists CV-6209 and WEB-2086 blocked PAF stimulated phosphorylation. This indicated structurally specific and PAF receptor dependency of this response. These results provide direct evidence that PAF stimulation of human platelets increased tyrosine kinase activity in pp60c-src immunoprecipitate.

Adult↗
PubMed1984

Monoclonal antibody-mediated immunoprecipitation of proteins from cells infected with Marek's disease virus or turkey herpesvirus.

The major immunogenic viral proteins of Marek's disease virus (MDV) and turkey herpesvirus (HVT) share antigenic determinants. The polyacrylamide gel electrophoresis pattern of five viral polypeptides immunoprecipitated with homologous convalescent chicken plasma was identical with the pattern obtained after immunoprecipitation with heterologous convalescent chicken plasma. A panel of monoclonal antibodies was used to identify MDV and HVT polypeptides. Sixteen monoclonal antibodies were positive in an immunofluorescence assay. However, only eight monoclonal antibodies immunoprecipitated a total of seven distinct viral proteins from MDV- and HVT-infected cells. Six of these monoclonals immunoprecipitated multiple viral proteins. None of the monoclonal antibodies recognized the common A antigen of MDV or HVT. Monoclonal antibodies immunoprecipitated three glycoproteins (100,000, 60,000, and 49,000 Da) that comigrate in polyacrylamide gels with three of the five common polypeptides obtained with the convalescent chicken plasma. In addition, a 79,000-Da protein was common to all MDV- and HVT-infected cells. Competition immunoprecipitation and peptide mapping by limited proteolysis confirmed that the three glycoproteins and the 79,000-Da protein contain MDV-HVT common epitopes. MDV-specific antigenic determinants were detected on the three remaining viral proteins (41,000, 38,000, and 24,000 Da).

Animals↗
PubMed2003

False interaction of syntaxin 1A with a Ca(2+)-activated K(+) channel revealed by co-immunoprecipitation and pull-down assays: implications for identification of protein-protein interactions.

The techniques of co-immunoprecipitation and immunocytochemical co-labelling are classically used to identify protein-protein interactions. We have used an antibody to the rat small conductance calcium-activated potassium channel subtype 1 (rSK1) to immunoprecipitate proteins from rat brain. A 35 kDa protein was recognized by two monoclonal antibodies to syntaxin 1 and a polyclonal antibody to syntaxin 1A, but not by antibodies to syntaxins 2, 3 or 4. These data suggested that syntaxin 1A is specifically associated with rSK1 in rat brain. A GST construct of the carboxyl terminus of rSK1 was able to pull-down syntaxin 1A from rat brain. Immunocytochemistry showed somatic labelling for both rSK1 and syntaxin 1A in acutely dissociated hippocampal CA1 neurons, confirming that these proteins could interact in vivo. However, control immunoprecipitations showed that antibodies to eight potassium channels could also immunoprecipitate syntaxin, even though some of these channels would not be expected to reside in the same subcellular compartment. Mock immunoprecipitations and pull-down assays showed that syntaxin 1 could directly interact with sepharose and agarose resins. Hence immunoprecipitation and pull-down assays do not provide evidence that syntaxin is specifically associating with a protein, placing doubt on a number of reported interactions with syntaxin 1A.

Animals↗
PubMed1990

Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gs alpha.

An antibody (RM) raised against the carboxyl-terminal decapeptide of the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Gs alpha) has been used to study the interaction of Gs alpha with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-gamma-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-gamma-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gs alpha protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gs alpha detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gs alpha protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Go alpha, Gi alpha, and G beta. The G beta protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of G beta was about the same in adenylate cyclase from preactivated membranes and from control membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed2000

Immunoprecipitation of high-affinity, guanine nucleotide-sensitive, solubilized mu-opioid receptors from rat brain: coimmunoprecipitation of the G proteins G(alpha o), G(alpha i1), and G(alpha i3).

Antibodies directed against the C-terminal and the N-terminal regions of the mu-opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the mu receptor. Two fusion proteins were constructed: One contained the 50 C-terminal amino acids of the mu receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating approximately 70% of solubilized rat brain mu receptors as determined by [3H][D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin ([3H]DAMGO) saturation binding. The material immunoprecipitated with both of the antisera was recognized as a broad band with a molecular mass between 60 and 75 kDa when screened in a western blot. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) had an EC50 of 0.4 nM in diminishing [3H]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to mu receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity-purified antibody was screened for the presence of G protein a subunits, it was determined that G(alpha)o, G(alpha)i1, G(alpha)i3, and to a lesser extent G(alpha)i2, but not G(alpha)s or G(alpha)q11, were coimmunoprecipitated with the mu receptor. Inclusion of GTPgammaS during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.

Animals↗
PubMed1989

Human cytomegalovirus RNAs immunoprecipitated by multiple systemic lupus erythematosus antisera.

The association of human cytomegalovirus (HCMV) RNAs with ribonucleoprotein particles that react with antibodies from patients with systemic lupus erythematosus was tested by immunoprecipitation with multiple patients' sera. A major late 2.8 kb RNA and several minor RNAs encoded by the HCMV long repeat region were immunoprecipitated from HCMV-infected cells by La, Ro and, much less abundantly, Sm autoimmune antisera. The exact location of these RNAs was determined by high resolution R-loop mapping and found to be between 0.8093 and 0.8189 map units. The 2.8 kb RNA is polyadenylated and associated with polysomes but does not appear to be spliced. Immunoprecipitation was not seen using normal or other autoimmune antisera. In addition, immunoprecipitation was specific to these RNAs in that other abundant HCMV RNAs were not immunoprecipitated. It was also found that the addition of increasing amounts of purified La antigen to infected cell lysates inhibited immunoprecipitation of the 2.8 kb RNA by La antiserum. The data suggest that specific HCMV RNAs may interact with cellular ribonucleoproteins known to be involved in post-transcriptional regulation of gene expression.

Antigen-Antibody Complex↗