FIG. 6.

Sin associates with and is constitutively phosphorylated by Fyn in resting cells. Sin is transiently dephosphorylated and dissociates from Fyn in response to TCR stimulation. (A) Jurkat cells (10⁷) stably transfected with vector alone or Sin-expressing plasmid were stimulated with OKT3 (5 μg/ml) at the indicated time points, and whole-cell extracts normalized for protein content were separated by SDS-PAGE and blotted with antiphosphotyrosine antibody. The blot was stripped and reprobed with anti-Sin to reveal total Sin levels (lower right panel). Similarly prepared extracts from vector or Sin-expressing cell lines were immunoprecipitated with Sin-specific antibody and first blotted with antiphosphotyrosine and then stripped and reprobed with Sin antibody (right panels). (B and C) Sin-expressing (5B1) or control Jurkat E6 cells (B) and 3 × 10⁷ thymocytes from normal mice (C) were left unstimulated or were stimulated with anti-TCR antibodies as shown. Cell extracts were immunoprecipitated with Sin-specific antibody or isotype-matched control IgG, and immune complexes were incubated in vitro in the presence of [³²P]ATP. Immune complexes were separated, proteins were transferred to nitrocellulose membrane, and membranes were exposed on film overnight. Subsequently, the filters were probed with Sin antibody to reveal total Sin levels. (D) Jurkat Tag cells (5 × 10⁶) transiently transfected with Sin alone and Sin plus Fyn or Lck were immunoprecipitated with anti-Sin antibody and processed as described above for panel A. The blots were sequentially probed with antiphosphotyrosine, Sin, and Fyn or Lck antibodies as shown. (E) Control or Sin-expressing Jurkat Tag cells were stimulated for the indicated times with OKT3 antibody, and extracts were immunoprecipitated with Fyn- or Lck-specific antibodies. Immune complexes were subjected to in vitro kinase assays in the presence of radioactive ATP and subsequently separated on SDS-PAGE. Blots were exposed on film and then probed sequentially with Sin and Fyn or Lck antibodies as shown. α-pY, antiphosphotyrosine; α-Sin, anti-Sin; pSin, phosphorylated Sin; WE, whole-cell extracts; iso, isoforms; α-Fyn, anti-Fyn; α-Lck, anti-Lck.