Preparation And Application Of Gα_q/11 Polyclonal Antibody

Heterotrimeric guanine-nucleotide-binding regulatory proteins belong to a protein family with GTP hydrolyase,which consist of Gα、Gβand Gγsubunit and have an important molecular switching role in signal transduction pathway.Gα_q/11 is a member of Gαprotein family widely in living things.So far,one of the best known functions for Gα_q/11 is to mediate Gα_q/11/PLCs signaling pathway.However,little research has been done on the biological function of Gα_q/11mediated cancer signaling pathway.To investigate the function of Gα_q/11 in the mediation of Gα_q/11 in cancer-related signaling,we need a large number of Gα_q/11 antibodies;for this purpose,we prepared Gα_q/11/11 antibody.According to the spatial structure and hydrophilic value of Gα_q/11,we choose the peptide（1-35AA and 104-168AA）of Gα_q/11/11 as the antigens.The recombinant expression vector was constructed and transformed into the E.coil Rosetta TM（DE3）to express antigen and purify,the results from coomassie brilliant blue method showed that we successfully obtained the target fusion protein.By immunological experiments on New Zealand white rabbits,the Gα_q/11anti-serum were obtained.By using affinity chromatography,we purified Anti-Gα_q/11 polyclonal antibody,and confirmed that the antibody has high specificity and the titer is 128000 by means of Western blotting and indirect ELISA assay.From the results of immunoprecipitate（IP）and Co-immunoprecipitate（CO-IP）,we found that the Gα_q/11/11 antibody can recognize the Gα_q/11 protein in a natural state,the silver staining experiment showed that proteins interacted with Gα_q/11 were coprecipitated.Through proteomics experiments,we screened out serious proteins interacting with Gα_q/11 by mass spectrometry.By analyzing in database clustering,we analyzed and classified the proteomics data,in order to preliminarily explore the function of Gα_q/11 in the signaling pathway of cancer cells,we took CD44 as the research candidate.Firstly,by using Co-IP,GST-pull down and immunofluorescent staining assay,we confirmed the interaction of Gα_q/11 and CD44.Secondly,in order to further investigate the effect of Gα_q/11 in the HA/CD44 signaling pathway,we observed the effect of Gα_q/11 on cell migration inducing by HA in HepG2 cells by transwell assay.We found that in the presence of HA,the migration ability of HepG2 cells was significa ntly improved,even under the condition of blocking CD44,the migration ability of HepG2 cells was unchanged.Additionally,we found the migration ability of HepG2 cells with silencing of Gα_q/11 was significantly decreased regardless of whether or not they were stimulated by HA,the similar results are shown even under the condition of blocking CD44.These results suggesting that Gα_q/11 mediates HA/CD44-induced HepG2 cell migration.In conclusion,this research provided a good tool for scientific research.By using this tool,we preliminarily investigated a novel function of Gα_q/11.We found that Gα_q/11 is involved in HA/CD44 signaling and impacts the capacity of cell migration,which are of some instructive significance for further research on the mediation of Gα_q/11/11 in cancer-related signaling and mechanisms of disease.