Figure 6. Effects of single treatments or combinations on proteins involved in the downstream signaling of the EGFR pathway and on cell cycle checkpoint-related proteins.

Western blot analysis showing protein levels in BxPC-3 and Capan-1 cells treated with G→E (panels A and B) and E→G (panel C and D). In all experimental groups, cells were treated with the single drugs (10 μM erlotinib, respectively 40 and 100 nM gemcitabine for BxPC-3 and Capan-1) or the combination and protein extracts were taken at the end of the last treatment (72 h) and 24 h after drug washout (96 h). Single treated samples were incubated with the drug at the same times scheduled for the combination (0–6 h for gemcitabine in the G→E group and 66–72 h in the E→G group; 24–72 h for erlotinib in the G→E group and 0–48 h in the E→G group). RAN and actin were used as loading controls.