Figure 2.

RIN1 coimmunoprecipitations from NIH 3T3 cells. (A) Cells expressing H-RAS^61L (+) or wild-type cells (−) were infected with a RIN1 expressing retrovirus (+) or were mock infected (−). Antibodies Y238 or Y259 were used to immunoprecipitation (IP) RAS. Y238* indictates preincubation with GST–RAS protein. Whole cell lysate (no IP) was included (lane 2). Samples were analyzed by Western blot using antibody to RAS (RAS) or RIN1C (RIN1). (B) Cells expressing H-RAS^61L and HA-RIN1C were lysed and subjected to electrophoresis directly (−) or immunoprecipitated with RAS antibody (Y238, Y238*, Y259) or 12CA5 antibody (αHA) and analyzed by Western blot as in A. (C) RIN1 and 14-3-3 copurify in immunoprecipitations. Cells expressing (+) or not expressing (−) HA-RIN1C were analyzed as lysates (lanes 1 and 2) or as an IP with the 12CA5 antibody and examined by Western blot using 14-3-3 or RIN1C antibody as probe.