Fig. 6. Cholesterol depletion induces increased tyrosine phosphorylation of the EGFR. (A) A431 cells and HEp-2 cells were pre-incubated with (+) or without (-) 10 mM MßCD for 15 minutes at 37°C, cooled with ice-cold PBS and further incubated with or without EGF in MEM with or without 10 mM MßCD for 15 minutes on ice. Whereas A431 cells were incubated with 0.1 nM EGF, HEp-2 cells were incubated with 1 nM EGF to achieve detectable ligand-induced phosphorylation. The cells were lysed and subjected to SDS-PAGE and western blotting, using antibodies to EGFR and PY1173. (B) A431 cells were pre-incubated with (+) or without (-) 1 µg/ml U18666A for 48 hours prior to incubation with or without EGF (0.1 or 1.0 nM) for 15 minutes on ice. To demonstrate differences in EGFR tyrosine phosphorylation (PY1173) upon incubation with 1 nM EGF, two different exposures of the blot are shown.