Biomedical subjects

Hans-Jürgen Thiesen

Publications and source records attributed to Hans-Jürgen Thiesen.

At least 19 recordsLinked to original sources

PubMed2006

Inhibitory effects of interferon-gamma on activation of rat pancreatic stellate cells are mediated by STAT1 and involve down-regulation of CTGF expression.

Pancreatic stellate cells (PSCs) are the main source of extracellular matrix proteins in pancreatic fibrosis, a pathological feature of chronic pancreatitis and pancreatic cancer. Interferon-gamma (IFN-gamma) is an antifibrotic cytokine, but how precisely it exerts its effects on PSCs is largely unknown. Here, we have focussed on the role of STAT1 as well as target genes of IFN-gamma signalling. Our data indicate that IFN-gamma regulates the expression of two autocrine mediators of PSC activation, connective tissue growth factor and endothelin-1, in a transforming growth factor-beta1-antagonistic manner. STAT1 overexpression under the control of a tetracycline-dependent promoter revealed a close correlation between STAT1 expression and activation, the biological effects of IFN-gamma (growth inhibition, induction of apoptosis), and target gene expression. Our data further support the hypothesis that IFN-gamma interferes with stellate cell activation in the pancreas and suggest activated STAT1 as an inductor of a quiescent PSC phenotype.

Animals↗

PubMed2006

Fine mapping of collagen-induced arthritis quantitative trait loci in an advanced intercross line.

The generation of advanced intercross lines (AIL) is a powerful approach for high-resolution fine mapping of quantitative trait loci (QTLs), because they accumulate much more recombination events compared with conventional F2 intercross and N2 backcross. However, the application of this approach is severely hampered by the requirements of excessive resources to maintain such crosses, i.e., in terms of animal care, space, and time. Therefore, in this study, we produced an AIL to fine map collagen-induced arthritis (CIA) QTLs using comparatively limited resources. We used only 308 (DBA/1 x FVB/N)F11/12 AIL mice to refine QTLs controlling the severity and onset of arthritis as well as the Ab response and T cell subset in CIA, namely Cia2, Cia27, and Trmq3. These QTLs were originally identified in (DBA/1 x FVB/N)F2 progeny. The confidence intervals of the three QTLs were refined from 40, 43, and 48 Mb to 12, 4.1, and 12 Mb, respectively. The data were complemented by the use of another QTL fine-mapping approach, haplotype analysis, to further refine Cia2 into a 2-Mb genomic region. To aid in the search for candidate genes for the QTLs, genome-wide expression profiling was performed to identify strain-specific differentially expressed genes within the confidence intervals. Of the 1396 strain-specific differentially expressed genes, 3, 3, and 12 genes were within the support intervals of the Cia2, Cia27, and Trmq3, respectively. In addition, this study revealed that Cia27 and Trmq3 controlling anti-CII IgG2a Ab and CD4:CD8 T cell ratio, respectively, also regulated CIA clinical phenotypes.

Animals↗

PubMed2006

Robust computational reconstitution - a new method for the comparative analysis of gene expression in tissues and isolated cell fractions.

BACKGROUND: Biological tissues consist of various cell types that differentially contribute to physiological and pathophysiological processes. Determining and analyzing cell type-specific gene expression under diverse conditions is therefore a central aim of biomedical research. The present study compares gene expression profiles in whole tissues and isolated cell fractions purified from these tissues in patients with rheumatoid arthritis and osteoarthritis. RESULTS: The expression profiles of the whole tissues were compared to computationally reconstituted expression profiles that combine the expression profiles of the isolated cell fractions (macrophages, fibroblasts, and non-adherent cells) according to their relative mRNA proportions in the tissue. The mRNA proportions were determined by trimmed robust regression using only the most robustly-expressed genes (1/3 to 1/2 of all measured genes), i.e. those showing the most similar expression in tissue and isolated cell fractions. The relative mRNA proportions were determined using several different chip evaluation methods, among which the MAS 5.0 signal algorithm appeared to be most robust. The computed mRNA proportions agreed well with the cell proportions determined by immunohistochemistry except for a minor number of outliers. Genes that were either regulated (i.e. differentially-expressed in tissue and isolated cell fractions) or robustly-expressed in all patients were identified using different test statistics. CONCLUSION: Robust Computational Reconstitution uses an intermediate number of robustly-expressed genes to estimate the relative mRNA proportions. This avoids both the exclusive dependence on the robust expression of individual, highly cell type-specific marker genes and the bias towards an equal distribution upon inclusion of all genes for computation.

Arthritis, Rheumatoid↗

PubMed2006

Characterization of ischemia/reperfusion-induced gene expression in experimental pancreas transplantation.

BACKGROUND: The aim of this study was to identify genes that are differentially expressed in the early period after pancreatic cold ischemia/reperfusion (I/R) injury. METHODS: Grafts of isogeneic rat pancreaticoduodenal transplantation were subjected to different preservation solutions and cold ischemia times (CITs): University of Wisconsin (UW), 6-hour CIT; UW, 18-hour CIT; and physiologic saline solution, 6-hour CIT. Animals that did not receive transplants served as controls. At 2-hour reperfusion, grafts were removed and pancreatic RNA was isolated, pooled, and hybridized to Affymetrix RG-U34A arrays. Quantitative reverse-transcription polymerase chain reaction was used to confirm the results of microarray technology. RESULTS: A total of 49 genes were consistently upregulated (more than threefold) in all three groups of transplant recipient animals. Prominent genes include transcription factors; cytoskeletal factors; heat-shock proteins (e.g. Hsp27, Hsp90); molecules involved in inflammation (e.g. PAPIII), immunology, signal transduction, and translation; and genes that have not been associated with I/R injury so far (e.g. Best5). Messenger RNA levels of some genes were exclusively downregulated in response to the different conditions applied to the pancreatic grafts: Cybb, Reg3a, Per2, BMAL1, MAP, and Isl2. CONCLUSIONS: These results provide new insight in I/R-induced gene expression after experimental pancreas transplantation. The reported upregulation of heat shock proteins, Best5, and PAPIII may play a pathologic role in pancreatic cold I/R injury and could therefore provide a promising perspective for further investigations.

Animals↗

PubMed2006

Cis- and trans-acting gene regulation is associated with osteoarthritis.

Osteoarthritis (OA) is a complex disease of the skeleton and is associated with aging. Both environmental and genetic factors contribute to its pathogenesis. We set out to identify novel genes associated with OA, concentrating on regulatory polymorphisms allowing for differential expression. Our strategy to identify differentially expressed genes included an initial transcriptome analysis of the peripheral blood mononuclear cells of six patients with OA and six age-matched healthy controls. These were screened for allelic expression imbalances and potentially regulatory single-nucleotide polymorphisms (SNPs) in the 5' regions of the genes. To establish disease association, disparate promoter SNP distributions correlating with the differential expression were tested on larger cohorts. Our approach yielded 26 candidate genes differentially expressed between patients and controls. Whereas BLP2 and CIAS1 seem to be trans-regulated, as the absence of allelic expression imbalances suggests, the presence of allelic imbalances confirms cis-regulatory mechanisms for RHOB and TXNDC3. Interestingly, on/off-switching suggests additional trans-regulation for TXNDC3. Moreover, we demonstrate for RHOB and TXNDC3 statistically significant associations between 5' SNPs and the disease that hint at regulatory functions. Investigating the respective genes functionally will not only shed light on the disease association but will also add to the understanding of the pathogenic processes involved in OA and may point out novel therapeutic approaches.

Adult↗

PubMed2006

Identification and management of fetuses at risk for, or affected by, congenital heart block associated with autoantibodies to SSA (Ro), SSB (La), or an HsEg5-like autoantigen.

The congenital heart block (CHB), diagnosed in structurally normal hearts, is strongly associated with, if not caused by, maternal SSA/SSB antibodies (Abs). It develops between 16 and 24 weeks' gestation, coincidentally with the increased transplacental IgG passage, and a window of unique cardiac vulnerability. Less is known about rare CHB cases in which neither cardiac malformations nor SSA/SSB Abs are detectable. We report on four pregnant women: patient 1 at high CHB risk (owing to Sjögren's syndrome (SS) and recurrent pregnancy losses), and patients 2-4 with already established CHB (aggravated by hydrops in patient 2). Abs were found directed to SSA/SSB (patients 1-3) or to an HsEg5-like autoantigen instead (patient 4). During preventive immunoadsorption (IA) from week 19 throughout (patient 1), or therapeutic IA (plus dexamethasone), commenced at week 25 (patient 2), SSA Ab levels decreased per session by 47+/-7 or 80+/-16%, respectively, and hydropic changes resolved. Patient 1 delivered a healthy boy, while patients 2-4 gave birth to CHB-affected children at need for permanent pacing. The irreversibility of complete CHB may justify (a) early ANA screening in all pregnancies (thereby also considering specificities as anti-HsEg5), and (b) preventive immmunoadsorption in high-risk pregnancies (before/during the critical cardiac development phase). This implies controversy, because factors converting risk to disease (in only approximately 2%) are unknown, and prospective randomized treatment studies are not available, given the rarity of CHB.

Autoantibodies↗

PubMed2006

Rheumatoid arthritis, a complex multifactorial disease: on the way toward individualized medicine.

With the availability of the human genome sequence and those of related species like chimpanzee, mouse, and rat, data driven research for tackling the molecular grounds of rheumatoid arthritis (RA), a multifactorial polygenic disease, can be considered a realistic challenge to the scientific community. A comprehensive research strategy is presented enabling the integration of multiple research efforts on studying autoimmunity by so called systems biology approaches. An integrative scientific concept is discussed of how to unravel molecular mechanisms of complex diseases by making use of state-of-the-art methodologies in functional and comparative genomics. A continuous interchange of data-driven and hypothesis-driven research is adjoined to determine the nature of rheumatic diseases with autoimmune background. Instead of studying single genes and proteins, RNA and protein microarray profiles are currently obtained in numerous research projects producing read-outs termed gene signatures rather than DNA and/or protein markers. A comprehensive study of the RNA, protein, and metabolite regimes is undertaken that eventually will lead to a "holistic" view of how all respective molecules, pathways and cells themselves interact with each other. Some of the above mentioned research aspects have already been studied by the authors, hopefully leading to new diagnostics and therapeutics in the future.

Animals↗

PubMed2005

Cytoskeletal rearrangements in synovial fibroblasts as a novel pathophysiological determinant of modeled rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory disease with a high prevalence and substantial socioeconomic burden. Despite intense research efforts, its aetiology and pathogenesis remain poorly understood. To identify novel genes and/or cellular pathways involved in the pathogenesis of the disease, we utilized a well-recognized tumour necrosis factor-driven animal model of this disease and performed high-throughput expression profiling with subtractive cDNA libraries and oligonucleotide microarray hybridizations, coupled with independent statistical analysis. This twin approach was validated by a number of different methods in other animal models of arthritis as well as in human patient samples, thus creating a unique list of disease modifiers of potential therapeutic value. Importantly, and through the integration of genetic linkage analysis and Gene Ontology-assisted functional discovery, we identified the gelsolin-driven synovial fibroblast cytoskeletal rearrangements as a novel pathophysiological determinant of the disease.

Animals↗

PubMed2005

Inhibition of lymphocyte apoptosis by pancreatic stellate cells: impact of interleukin-15.

There is growing evidence that pancreatic stellate cells (PSCs) produce cytokines and take part in the regulation of inflammatory processes in the pancreas. IL-15 inhibits apoptosis of various cell populations. This study was performed to investigate whether PSCs produce IL-15 and thereby can affect lymphocytes. Primary PSCs were isolated from the rat pancreas using density gradient centrifugation. mRNA expression of IL-15 was demonstrated by RT-PCR, and IL-15 protein was analyzed by immunoblotting. Lymphocytes obtained from rat mesenterial lymph nodes were cocultured with in vitro activated PSCs. Apoptosis has been quantified by the binding of annexin V-FITC with a flow cytometer. Proliferation was monitored using [3H]thymidine incorporation. PSCs express two splice variants of IL-15. The protein was detectable only in cell lysates but not in the cell culture supernatant. Cocultivation of lymphocytes with PSCs and IL-15 inhibited spontaneous lymphocyte apoptosis, and this effect was reduced by an anti-IL-15 antibody. Lymphocytes induced vice versa the proliferation and collagen production of PSCs. The inhibition of spontaneous lymphocyte apoptosis in cocultures with PSCs was at least partially mediated by cell-bound IL-15. This effect and the stimulation of PSCs by lymphocytes may lead to a circulus vitiosus, resulting in the persistence of inflammatory processes and the development of fibrosis during chronic pancreatitis.

Animals↗

PubMed2005

Collection of soluble variants of membrane proteins for transcriptomics and proteomics.

The existence of a soluble splice variant for a gene encoding a transmembrane protein suggests that this gene plays a role in intercellular signalling, particularly in immunological processes. Also, the absence of a splice variant of a reported soluble variant suggests exclusive control of the solubilisation by proteolytic cleavage. Soluble splice variants of membrane proteins may also be interesting targets for crystallisation as their structure may be expected to preserve, at least partially, their function as integral membrane proteins, whose structures are most difficult to determine. This paper presents a dataset derived from the literature in an attempt to collect all reported soluble variants of membrane proteins, be they splice variants or shedded. A list of soluble variants is derived in silico from Ensembl. These are checked on their presence in multiple organisms and their number of membranespanning regions is inspected. The findings then are confirmed by a comparison with identified proteins of a recent global proteomics study of human blood plasma. Finally, a tool to determine novel soluble variants by proteomics is provided.

Animals↗

PubMed2005

Global epithelial cell transcriptional responses reveal Streptococcus pyogenes Fas regulator activity association with bacterial aggressiveness.

The bacterial human pathogen Streptococcus pyogenes (group A streptococci, GAS) is able to adhere to, internalize into and cross-talk on multiple levels with its host cells. To gain insight into the Fas function in pathogenesis we used Affymetrix human genome DNA-arrays to measure temporal and global transcriptional responses of HEp-2 cells infected with M49 S. pyogenes wild-type bacteria and DeltafasX, an isogenic S. pyogenes two-component-signal-transduction system mutant. A modified stringent statistical analysis method identified a total of 86 HEp-2 cell genes as differentially transcribed upon infection over the investigated time course. Increased expression of genes encoding proteins involved in GAS host cell adherence and internalization (fibronectin, integrin-alpha5) was found as a common response. In contrast to earlier reports investigating other GAS serotype strains, Ras superfamily and RhoA pathways are exploited by M49 GAS, suggesting serotype specific interactions with the host cell cytoskeleton. Despite transcriptional induction, secreted IL-8 levels of deltafasX mutant infected cells were below those of non-infected cells, indicating an absence of Fas expression could be important for GAS tissue colonization and long-term intracellular persistence. Oppositely, activity of the S. pyogenes Fas-system apparently promotes high adherence and internalization rates, massive cytokine gene transcription and cytokine release, host cell apoptosis via a caspase-2 activation pathway, and cytotoxicity. Thus, the S. pyogenes Fas two-component signal transduction system could be involved in local tissue destruction and general bacterial aggressiveness towards host cells.

Apoptosis↗

PubMed2004

A proinflammatory role for Fas in joints of mice with collagen-induced arthritis.

Collagen-induced arthritis (CIA) is a chronic inflammatory disease bearing all the hallmarks of rheumatoid arthritis, e.g. polyarthritis, synovitis, and subsequent cartilage/bone erosions. One feature of the disease contributing to joint damage is synovial hyperplasia. The factors responsible for the hyperplasia are unknown; however, an imbalance between rates of cell proliferation and cell death (apoptosis) has been suggested. To evaluate the role of a major pathway of cell death - Fas (CD95)/FasL - in the pathogenesis of CIA, DBA/1J mice with a mutation of the Fas gene (lpr) were generated. The susceptibility of the mutant DBA-lpr/lpr mice to arthritis induced by collagen type II was evaluated. Contrary to expectations, the DBA-lpr/lpr mice developed significantly milder disease than the control littermates. The incidence of disease was also significantly lower in the lpr/lpr mice than in the controls (40% versus 81%; P < 0.05). However DBA-lpr/lpr mice mounted a robust immune response to collagen, and the expression of local proinflammatory cytokines such as, e.g., tumor necrosis factor alpha (TNF-alpha) and IL-6 were increased at the onset of disease. Since the contribution of synovial fibroblasts to inflammation and joint destruction is crucial, the potential activating effect of Fas on mouse fibroblast cell line NIH3T3 was investigated. On treatment with anti-Fas in vitro, the cell death of NIH3T3 fibroblasts was reduced and the expression of proinflammatory cytokines TNF-alpha and IL-6 was increased. These findings suggest that impairment of immune tolerance by increased T-cell reactivity does not lead to enhanced susceptibility to CIA and point to a role of Fas in joint destruction.

Animals↗

PubMed2004

Overexpression of glial cell line-derived neurotrophic factor induces genes regulating migration and differentiation of neuronal progenitor cells.

The glial cell line-derived neurotrophic factor (GDNF) is involved in the development and maintenance of neural tissues. Mutations in components of its signaling pathway lead to severe migration deficits of neuronal crest stem cells, tumor formation, or ablation of the urinary system. In animal models of Parkinson's disease, GDNF has been recognized to be neuroprotective and to improve motor function when delivered into the cerebral ventricles or into the substantia nigra. Here, we characterize the network of 43 genes induced by GDNF overproduction of neuronal progenitor cells (ST14A), which mainly regulate migration and differentiation of neuronal progenitor cells. GDNF down-regulates doublecortin, Paf-ah1b (Lis1), dynamin, and alpha-tubulin, which are involved in neocortical lamination and cytoskeletal reorganization. Axonal guidance depends on cell-surface molecules and extracellular matrix proteins. Laminin, Mpl3, Alcam, Bin1, Id1, Id2, Id3, neuregulin1, the ephrinB2-receptor, neuritin, focal adhesion kinase (FAK), Tc10, Pdpk1, clusterin, GTP-cyclooxygenase1, and follistatin are genes up-regulated by GDNF overexpression. Moreover, we found four key enzymes of the cholesterol-synthesis pathway to be down-regulated leading to decreased farnesyl-pyrophospate production. Many proteins are anchored by farnesyl-derivates at the cell membrane. The identification of these GDNF-regulated genes may open new opportunities for directly influencing differentiation and developmental processes of neurons.

Animals↗

PubMed2004

Generation and characterization of immortalized rat pancreatic stellate cells.

Pancreatic stellate cells (PSCs) are involved in, among other things, the pathogenesis of pancreatic fibrosis. Here, we present the generation of immortalized PSCs 7 and 14 days after isolation by retroviral gene transfer of the SV40 large T antigen encoding region. Propagated cell lines [large T immortalized cells (LTC)-7, LTC-14] retained characteristics of primary cells in terms of morphology, responsiveness to mediators regulating cellular functions such as proliferation, and expression profile of a number of investigated genes. Whereas LTC-14 kept the morphological features of the differentiation status of the primary cells they were made of, LTC-7 appeared similar to an earlier stage. Thus the established cell lines represent a versatile tool to investigate various aspects of PSC biology.

Animals↗

PubMed2004

Differential proteome analysis and mass spectrometric characterization of germ line development-related proteins of Caenorhabditis elegans.

Proteome maps and differences of protein patterns of the synchronized larval stage L4 of the temperature-sensitive Caenorhabditis elegans (C. elegans) glp-1 mutant (e2144ts) were investigated after cultivation at 15 degrees C (developing a normal phenotype) or 25 degrees C (developing a mutated phenotype) by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. From the 183 identified protein spots six proteins were found differently expressed. The Vit-6 vitellogenin (CE28594), the hypothetical 17.2 protein (CE25224), the hypothetical 17.4 protein (CE16999), and the heat shock protein 16 kDa (CE14249) were more abundant when growing worm cultures at 25 degrees C. By contrast, the nucleoside diphosphate kinase (CE09650) was found increased at 15 degrees C. Most notably, the eukariotic initiation factor 5A-1 (CE00503), highly abundant at 15 degrees C, was not present in cultures grown at 25 degrees C. Its absence at 25 degrees C can not be attributed to lack of the enzymatic machinery that is necessary for hypusinylation. Instead, a direct downstream effect of the lack of functionality of GLP-1 may cause the expression of this protein. The yolk proteins 115 kDa and 88 kDa were attributed by mass spectrometric protein structure analysis as C-terminal and N-terminal fragments of the Vit-6 vitellogin protein (CE28594), respectively. The cleavage site between both derivatives was located between R764 and A768. A conflict in the database sequences at amino acid positions 1622 and 1623 of vitellogenin-6 was solved by mass spectrometric sequence analysis. The combination of 2-DE with mass spectrometry enabled the identification of mutation-associated differences on somatic gonadal cell and germ line cell development-associated proteins.

Amino Acid Sequence↗

PubMed2004

Gene-expression profiling of the early stages of MOG-induced EAE proves EAE-resistance as an active process.

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) is a well-established animal model of multiple sclerosis (MS) in rodents. It reflects the wide spectrum of disease pathology and serves as a valuable tool for studying the pathogenesis and for testing new therapies of MS. In order to identify genes responsible for resistance to and modulation of the disease, we compared the mRNA expression profile of more than 12,000 genes by DNA microarray technique in lymph nodes of the highly EAE-susceptible mouse strain C57Bl/6 (B6) and the resistant strain C57Bl/10.S (B10). The disease onset in B6 mice was day 15. We identified 84 genes that were up-regulated more than two-fold in B10 mice compared to vehicle-treated controls, whereas only two genes were up-regulated in B6 mice after 7 and 15 days post-immunization (p.i.), respectively. We were able to match five up-regulated genes in B10 mice to known quantitative trait loci (QTLs), which control for EAE susceptibility. Only 17, respectively 5, genes were down-regulated at both time points in B10 and B6 mice. Tests for immunoreactivity to MOG (T cell proliferation and interferon-gamma (IFN-gamma) secretion) revealed no stronger immune response in B6 compared to B10 mice supporting the hypothesis of an immunosuppressive effect as a target to prevent EAE in the B10 mice. We conclude that resistance to EAE (and possibly to MS) is an active process mediated by multiple genes up-regulated in peripheral lymphatic organs of resistant animals. Thus, monitoring of the expression of these new candidate genes may serve as a tool for the disease progression and the pharmaceutical treatment.

Animals↗

PubMed2004

Proteome analysis reveals disease-associated marker proteins to differentiate RA patients from other inflammatory joint diseases with the potential to monitor anti-TNFalpha therapy.

New experimental approaches of molecular medicine such as transcriptome and proteome analysis have been implemented in rheumatology research. Two-dimensional gel electrophoresis in combination with mass spectrometry was used to visualize and to identify proteins in synovial fluid (SF) and plasma samples from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The small calcium binding protein S100A9 (MRP14) was identified as a discriminatory marker protein in SF by global proteomic analysis. To confirm these results and to examine the reproducibility and the applicability as a diagnostic marker, levels of the S100A8 (MRP8)/A9 (MRP14) heterocomplex in plasma and in synovial fluid were validated from patients with RA, OA, and other inflammatory joint diseases using enzyme immunoassay techniques. It was found that plasma levels of the S100A8/A9 heterocomplex correlate well with levels in SF, and hence, determination of plasma levels can be used to distinguish RA patients from patients with other inflammatory joint diseases, as well as from OA patients and controls. Initial studies on RA patients also indicate that plasma levels of the S100A8/A9 heterocomplex are a useful marker in monitoring anti TNFalpha therapy.

ATP-Binding Cassette Transporters↗

PubMed2004

From transcription profile to expression: the signaling repertoire of the SK-N-MC neuroepithelioma cell-line.

SK-N-MC neuroepithelioma cells are routinely cultured and widely used as a model system in biochemical and pharmacological experiments. To clarify the gene expression patterns of SK-N-MC cells with respect to G protein-coupled receptors and signaling network components, we describe in this report the transcription profile of the cell line. Following the traditional pathway from genome to proteome, selected examples are further examined at the level of protein expression and by functional assays. cRNA targets derived from total RNA extracts were hybridized to Affymetrix Human Genome U133A GeneChip arrays, and the data were analyzed and grouped according to functional aspects. Results obtained for neuropeptide Y (NPY) Y1, Y5, and orexin Ox1 receptors were confirmed by RT-PCR. It is surprising that we found the presence of both NPY receptor subtypes and the absence of the orexin receptor at the mRNA level. Receptor-binding experiments confirmed NPY binding of the Y1 receptor in the nanomolar range but gave no evidence for high expression levels of Y5 receptor subtypes on the cell surface. Protein expression was assayed with immunoblots by using antibodies directed against selected Galpha protein subunits. The presence of at least Galphas, Galphai3, and Galphai2 subunits was indicated.

Binding Sites↗