Figure 5

PKCß inhibition reduced constitutive activation of Src, decreased phosphorylation of c-Met and ErbB3, and had downstream effects on Akt and GSK-3ß in both Calu3 and H1975 cells. (A) PCKß inhibitor enzastaurin demonstrates that PKCß is required for Src activation. Concentrations of enzastaurin from 0–20 μM were added to unstimulated, serum-starved cells for 3.5 hours before lysates were prepared for Western blotting. Multiple blots were probed with antibodies against phospho-pan PKC, phospho-PKCα,ß, phospho-GSK-3ß, Akt and actin. (B) PKC inhibitor blocks constitutive activation. Unstimulated, serum-starved Calu3 and H1975 cells were cultured for 3.5 hours with 10 μM enzastaurin before lysates were prepared for Western blotting. Multiple blots were probed with antibodies against phospho-ErbB3 (Y-1289), phospho-c-Met (Y-1230,1234,1235), phospho-PKCα,ß, phospho-Src and actin. (C) Specificity of inhibition for PKCßII was confirmed by culture of unstimulated Calu3 cells with 80 μM PKCßII specific peptide inhibitor. Western blots were probed with anti-PKCβII antibodies, anti-phospho-Src (Y-416) and anti-actin.