Fig. 4. Crosslinked FcRII in U937 cells associates with Lyn and undergoes tyrosine phosphorylation in DRMs. (A) Tyrosine phosphorylation (PY) of proteins in OptiPrep gradient fractions. Cells were exposed to 1 mM HMA, 100 µM piceatannol, 15 µM PP1 or 5 mM CDX or left untreated (control) before crosslinking of FcRII with biotin-labeled IV.3 mouse anti-FcRII and goat anti-mouse IgG. For the analysis, 12 µl of gradient fractions 1-2 and only 4 µl of fraction 6 were used for a clearer visualization of proteins. Molecular mass standards are shown in kDa. An arrowhead marks the 40 kDa phosphoprotein corresponding to FcRII. Dots indicate proteins whose phosphorylation was significantly reduced by piceatannol. (B) Immunoprecipitation of FcRII from the gradient fractions (200 µl each). Immunoprecipitates were immunoblotted to simultaneously detect biotin-labeled anti-FcRII, reflecting the presence of the receptor (upper panel) and tyrosine-phosphorylated proteins (PY) (middle panel). An arrowhead points to FcRII; small arrows point to a doublet of 53/56 kDa phosphoproteins. The blots from the middle panel were reprobed with rabbit anti-Lyn (lower panel). The data represent one out of three experiments.