G protein gamma subunits coimmunoprecipitated with antibodies against alpha subunits: identification of major isoforms in cultured cells by silver stain and immunoblotting with conventional transfer procedure.

Abstract

The betagamma subunits of G proteins were coimmunoprecipitated with antibodies against various alpha subunits, and analyzed by silver stain and immunoblotting with conventional transfer procedure and membrane-blocking buffer containing 2% BSA. Multiple isoforms of gamma were coimmunoprecipitated with no significant difference in form or ratio among the antibodies against alpha subunits used, suggesting antibodies against any alpha subunit could coimmunoprecipitate all forms of gamma. Therefore, this method was applicable to analyze gamma subunits in various cells, especially to clarify what forms of gamma subunits are major components. The major isoforms were: gamma5 in C6, NG108-15, HeLa, HEK293, and F9 cells; gamma12 in Swiss 3T3 and BRL-3A cells; and gamma3 in PC12 cells. In addition to most gamma subunits identified, unidentified gamma subunits were present in PC12, NG108-15, and BRL-3A cells. Furthermore, the method was applied to examine changes of isoforms of gamma during differentiation of HL-60 cells. Undifferentiated cells mainly contained gamma5, but retinoic acid treatment of cells replaced most gamma5 with gamma2. Thus, this method is useful to determine the major isoforms which seem to be the more important in cells.