Fig. 6.

Src activation is essential for the CCh-induced EGFR transactivation cascade, resulting in cytoprotection against apoptotic challenges. A and B, HSG cells were exposed to CCh (100 μM) for the indicated times. C and D, HSG cells were pretreated with or without 4-DAMP (1 μM) for 30 min and then were exposed to CCh (100 μM) for 2 min. A and C, the phosphorylated (p) and total (t) Src levels were analyzed through immunoblotting. B and D, cell lysates were prepared and used for immunoprecipitation (IP) with antibody to total EGFR. The levels of c-Src protein coimmunoprecipitated with EGFR and the total amounts of EGFR were visualized through immunoblotting (IB). E, HSG cells were pretreated with or without PP2 (5 μM) for 30 min and then were exposed to CCh (100 μM) for 2 min. The phosphorylated and total EGFR, ERK, and Akt levels were analyzed through immunoblotting. Quantification of the band density was performed through densitometric scanning of each band by using National Institutes of Health ImageJ software. F, HSG cells were pretreated with or without PP2 (5 μM) for 30 min. The cells then were treated with or without 100 μM CCh, in the absence or presence of TNFα (50 ng/ml)/IFNγ (10 ng/ml), and were incubated for 24 h. Caspase 3/7 activity was indicated by luminescence activity, as described under Materials and Methods. Values represent the mean ± S.D. of three cultures. **, p < 0.01, values differ significantly (t test).