T5A7-induced phosphorylation of Lyn and p38 MAPK in DIM.

DIM isolated from neutrophils was kept on ice in a polypropylene tube (resting) or placed in normal IgM (normal IgM) or anti-LacCer IgM T5A7–coated dishes (anti-LacCer IgM) at 4°C for 15 hours. Then the mixtures were incubated with [γ-³²P]-ATP in the absence or presence of 100 nM PP1 for 5 minutes at 37°C, followed by trichloroacetic acid (TCA) precipitation. (A) The TCA precipitate was solubilized in lysis buffer B, immunoprecipitated with mouse anti-Lyn mAb, and subjected to SDS-PAGE, electroblotting transfer, and autoradiography (autoradiogram). To evaluate the recovery of Lyn, the blotted membrane was probed with rabbit anti-Lyn IgG (immunoblot). (B) The TCA precipitate was solubilized in RIPA buffer and immunoprecipitated with mouse anti-Lyn mAb. The immunoprecipitate was washed with RIPA buffer and subjected to SDS-PAGE, electroblotting, and autoradiography. (C) The TCA precipitate was solubilized in RIPA buffer and immunoprecipitated with mouse anti-pp38 MAPK mAb and subjected to SDS-PAGE, electroblotting, and autoradiography. In all 3 panels, locations of molecular markers are indicated on the left.