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. 1999 May 25;96(11):6211–6216. doi: 10.1073/pnas.96.11.6211

Figure 4.

Figure 4

Analysis of SH3-containing proteins for their interaction with CMS. (A) GST-fusion peptides coupled to glutathione Sepharose beads were incubated with lysates of 293T cells expressing flag-tagged CMS. Precipitates were analyzed by SDS/PAGE and WB with anti-flag antibody. (B) In vivo interaction of CMS with the tyrosine kinases Fyn and Yes. Lysates of 293T cells expressing flag-tagged CMS-TH or control vector were incubated with anti-Fyn and anti-Yes antibody coupled to protein A/G agarose beads. Immunoprecipitates (IP) were analyzed in SDS/PAGE and WB with anti-flag antibody. (C) Kinase activity is associated with CMS. Lysates of 293T cells expressing flag-tagged CMS or vector control were incubated with anti-flag antibody coupled to protein A/G agarose, and immunoprecipitates (IP) were subjected to a kinase assay. (D) Tyrosine phosphorylation of CMS. 293T cell lysates expressing flag-tagged CMS together with the indicated tyrosine kinases [wild type (WT) or kinase dead (KD)] were immunoprecipitated (IP) with antiphoshotyrosine antibody (4G10) and analyzed by WB with antiphosphotyrosine antibody and anti-CMS polyclonal serum. Total cell lysates were analyzed for expression of the transfected plasmids (not shown).