Journal of General Virology

Fig. 3. (A)–(B) StpB is phosphorylated by c-Src in vivo and in vitro. The vector pcDNA3 (lanes 1, 4, 7) and plasmids encoding AU–StpA (2, 5, 8) or AU–StpB (3, 6, 9) were transfected into COS cells either alone (lanes 1–3) or together with pFJ-Src (4–6) or pFJ-Src L295 (7–9). (A) Cell lysates (10 µg per lane) were separated by SDS–PAGE and subjected to an immunoblot with anti-phosphotyrosine antibody (upper panel). The membrane was cut horizontally and reprobed with MAb EC10 to detect c-Src (middle panel) or with the AU1-specific antibody to detect recombinant StpA and StpB (lower panel). (B) The same cell lysates (250 µg per sample) were used for AU1-specific immunoprecipitation followed by an in vitro kinase reaction, separation by SDS–PAGE and autoradiography (2 h exposure). Sizes of molecular mass markers are given in kDa on the left. Arrowheads indicate the position of c-Src; brackets span the position of StpA and StpB proteins. (C)–(D) Association of StpA and StpB with deletion mutants of c-Src. (C) AU–StpA (StpA), wild-type c-Src (wt) or c-Src deletion mutants lacking the SH3 (SrcSH3) or SH2 (SrcSH2) domains were expressed in COS cells either alone or in combinations as indicated. Cells were lysed and, after immunoprecipitation with SRC2 antiserum, proteins were separated by SDS–PAGE and co-precipitated proteins were detected by immunoblot with anti-AU1 antibodies (upper panel). Protein expression was confirmed by immunoblot analysis of cell lysates (middle and lower panel). Brackets indicate epitope-tagged proteins identified by the anti-AU1 MAb. Arrowheads indicate endogenous and recombinant Src proteins recognized by SRC2 antibody. (D) The same experiment was performed with AU–StpB (StpB). Sizes of molecular mass markers are given in kDa. *, Background band related to protein A–Sepharose.

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