Figure 11.

Interaction of TRIM with intracellular signaling molecules. (A) 10⁷ HPB-ALL cells per lane were activated for 2 min by cross-linking their CD3 and CD4 molecules using biotinylated Abs. Postnuclear lysates of the stimulated cells were incubated with the recombinant GST–SH2 domain fusion proteins depicted (top of lanes). Precipitates were run on reducing 14% SDS-PAGE and subjected to TRIM Western blotting. PI3K, PI3-kinase. N + C, NH- and COOH-terminal SH2 domains of the corresponding fraction. (B) HPB-ALL cells were either left unstimulated or were stimulated for 2 min by CD3 × CD4 cross-linking as described above. Lysates corresponding to 5 × 10⁵ cells were analyzed by anti-PTYR Western blotting (left), and the remaining material was subjected to TRIM immunoprecipitation (IP). Precipitates were run on reducing 12% SDS-PAGE and subjected to Western blotting using anti-Grb2 or anti-p85 mAbs (right). Note that the Grb2 blot had to be overexposed (five times longer compared with the p85 blot) to visualize coprecipitation of Grb2.