Fig. 5. Wortmannin inhibits the interaction between p120 Ras GAP and Rab5. (A) The effects of wortmannin on the membrane association of p120 Ras GAP. BT20 cells were subcellularly fractionated into soluble fraction (S100) and particulate fraction (P100) after treatment with wortmannin (100 nM) and EGF (30 min) as indicated. The subcellular fractions were subjected to immunoblot analysis with anti-p120 Ras GAP antibody as described in Methods. (B) The effects of wortmannin on the association of Rab5 and p120 Ras GAP. BT20 cells were treated with wortmannin for 15 min or not treated, and then stimulated with EGF for 30 min or not stimulated. Cell lysates were subjected to immunoprecipitation with rabbit anti-p120 Ras GAP antibody, followed by protein A–Sepharose. Immunoprecipitates were subjected to immunoblot analysis with mouse anti-Rab5 or anti-Rabaptin5 antibody.