The effects of Y27632 on the internalization of the EGFR in Panc1 pancreatic cancer cells. (A) Panc1 cells were treated with the indicated concentrations of Y27632 or vehicle for 1 h at 37°C, and then labeled for 15 min at 37°C with anti-EGFR antibodies which recognize the extracellular domain of the EGFR. The cells were then exposed to 30 ng/ml of EGF for 10 min at 37°C. To observe only the cell surface EGFR that remained on the plasma membrane, these cells were not permeabilized with Triton X-100. They were then exposed to Alexa Fluor 488®-conjugated donkey anti-goat IgG antibodies and DAPI for 1 h. Representative cells examined by fluorescence microscopy are shown. (B) Green fluorescence signals from the remaining cell surface EGFR were measured using the NIH image software program, and expressed as the % of the control (compared with untreated control cells).