FIG. 1.

HRG induces Stat3, Jak1, Jak2, and c-Src tyrosine phosphorylation. Cultures of C4HD (A to C) and T47D (D and E) cells were treated with HRG for the indicated times. Cell lysates (50 μg) were analyzed by Western blotting with phosphotyrosine 705 Stat3, phosphotyrosine 1022/1023 Jak1, and phosphotyrosine 1007/1008 Jak2 antibodies. Membranes were stripped and hybridized with total protein antibodies. c-Src activation was studied as described in Materials and Methods in cells treated and untreated with PP2 before HRG treatment. As a control, lysates were immunoprecipitated with normal mouse serum (NMS). Identical aliquots of each immunoprecipitate were subjected to immunoblot analysis with total c-Src antibody as a loading control. Phospho-protein bands underwent densitometry, and values were normalized to total protein bands. Data analysis showed that the increases in Stat3, Jak, and c-Src phosphorylation in cells treated for 10 min with HRG compared with the levels in untreated cells and the inhibition of HRG-induced c-Src phosphorylation levels caused by PP2 were significant (P < 0.001). Experiments shown were repeated five times with similar results. W, Western blot; IP, immunoprecipitation.