Fig. 3. Cholesterol depletion inhibits activation of the ERK/MAPK pathway by EphB1. (A) CHO-EphB1 cells were incubated with 0 (Fig. A1,2) or 10 mM (Fig. A3,4) ß-cyclodextrin (ß-CD) for 60 minutes, when cells were prepared for electron microscopy as described under Materials and Methods. Transmission electron microscopy (Fig. A, panels 1 and 3) and scanning electron microscopy (Fig. A, panels 2 and 4) reveal caveolae structures on the surface of the untreated CHO-EphB1 cells, indicated by arrowheads (insert in Fig. A1). Bars, 3 µm (panels 1,3), 12 µm (panels 2,4), and 5 µm (inserts in panels 2,4). A single cell is shown in the inserts shown in panels 2 and 4. (B,C) CHO-EphB1 cells were incubated with or without 10 mM ß-CD for 60 minutes, and then stimulated with 1-2 µg/ml ephrinB2 ligand for 30 minutes. (B) Co-immunoprecipitation (IP) of EphB1 receptor with antibody against caveolin-1 in CHO-EphB1 cells. The immunoprecipitates were resolved by SDS-PAGE and transblotted to PVDF membranes. Membranes were reciprocally immunoblotted with antibodies against HA or caveolin-1, respectively. (C) The state of ERK phosphorylation was determined by immunoblotting with a phosphospecific antibody against active ERK (P-Erk). Stripped membranes were then reblotted with antibody against ERK (Erk1/2). Relative phosphorylation levels are given as a ratio of phosphorylated protein expression to unphosphorylated protein expression. Protein expressions were analyzed using Image J software (http://rsb.info.nih.gov/ij/). Results are representative of at least three independent experiments.