Figure 2

Src Family Kinase (SFK) expression and activation. (A) Src-kinase inhibitor PP2, blocked phosphorylation of EGFR at Y-845 and Y-992 and downstream phosphorylations of Akt and Erk1,2. Calu3 cells were serum starved for 6 hours, then cultured with 10 μM PP2, 10 or 20 μM U0126, or an equivalent volume of DMSO for 1 hour with addition of recombinant EGF @ 1 μg/ml for the final 10 minutes; then lysates prepared for SDS-PAGE and Western blotting with indicated antibodies. (B) PP2 decreased viability of Calu3 cells in a concentration dependent manner. Calu3 cells were cultured in triplicate in 96-well plates, and serum starved for 8 hours before addition of the respective inhibitors with highest concentrations for PP2 @ 20 μM; LY29004 @ 40 μM; Erlotinib @ 20 μM; and DMSO @ 4 μl; then dilutions of one-half through seven serial titrations (7 → 1). After 70 hours, Calcein AM was added two hours before harvesting. (C) Distinct patterns of SFK activation were revealed in a quantitative Milliplex assay of unstimulated Calu3 and H1975 cell lysates. MFI (median fluorescence intensity) equals sample - background. (D) Expression of phosphorylated Lyn, Src, and an isoform of Fyn in Calu3 cell lysates were confirmed by Western blotting. Anti-phospho-Src (Y-416) immunoprecipitates were blotted and probed separately with anti-SFK member antibodies including Yes, Lyn, Fyn, Hck, and v-Src. Anti-Hck served as a specificity control in that no non-specific bands were observed in either the lysates or IP. (E) Lyn expression and phosphorylation were further confirmed by direct immunoprecipitation. Calu3 cell lysates were incubated with anti-phospho-Src (Y-417), anti-vimentin isotype control or no antibody. Duplicate immunoprecipitations were performed with recombinant protein A\G conjugated beads (PAG) (wells 2,5,7) or TrueBlot ® anti-light chain beads (TB, wells 4,6,8). Control immunoprecipitations demonstrated no extraneous bands near mw of SFKs, 58–66 kDa.