Stimulation of protein tyrosine phosphorylation via CD44. Freshly isolated human PBMC were stimulated by immobilized anti-CD44 MoAb as described in the Materials and Methods. At the indicated time points, cells were lysed and equivalent amounts of lysates were electrophoresed under reducing conditions, blotted, and probed for tyrosine phosphorylated proteins by PY20 followed by GAM-AP and ECL detection (upper panel). Lane 1, unstimulated cells; lanes 2 through 6, cells plated in CD44 MoAb-coated wells, lane 7, cells plated in control mouse IgG-coated wells (C). Equivalent protein loading in the wells was verified by Ponceau-S staining of the blots, as well as by probing for Lck and Fyn kinases (lower panels).