Fig. 4. Dominant-negative mutants of PI3-kinase and Rac1 inhibit MAPK activation induced by L1 clustering in B35 neuroblastoma cells. A, B35-L1 cells were incubated with mouse IgG or the L1 monoclonal antibody Neuro4 complexed with F(ab′)2fragments of anti-mouse IgG. Cell lysates (800 μg) were immunoprecipitated with anti-phosphotyrosine mAb (4G10), and the precipitates were incubated in an in vitro kinase reaction with phosphoinositides. Phospholipid products were separated by thin-layer chromatography (top). Error bars in the graph (bottom) indicate SEs based on three experiments; an asterisk denotes statistical significance (p < 0.05). B,Top row, B35-L1 cells transiently expressing HA–ERK2 and the dominant-negative (DN) mutants PI3-kinase (PI3K) Δp85, RhoA(19N), Rac1(17N), Cdc42(17N), FRNK, Fak(397F), or Ras(15A) were incubated with mouse IgG or the L1 monoclonal antibody Neuro4 complexed with F(ab′)2fragments of anti-mouse IgG. As a control, cells were exposed to 100 ng/ml EGF for 5 min. Cell extracts were immunoprecipitated with anti-HA antibodies and then subjected to SDS-PAGE and immunoblotting with anti-active MAPK antibody (p ERK2). Bottom row, The same nitrocellulose filters were stripped and reblotted with an antibody recognizing ERK2 protein. The experiments were performed three times with similar results. C,B35-L1 cells were incubated with normal mouse IgG or the L1 monoclonal antibody Neuro4 complexed with F(ab′)2 fragments of anti-mouse IgG for 3 and 8 min. GTP-loaded Rac was pulled down from the cell lysates (500 μg) by the addition of RBD–GST fusion protein conjugated to Sepharose beads, and the relative levels of Rac–GTP were evaluated by SDS-PAGE and immunoblotting with anti-Rac1 antibodies. D,Top, B35-L1 cells were exposed to the C3 toxin Rho inhibitor or the Ly294002 PI3-kinase inhibitor as described in Materials and Methods and then incubated with normal mouse IgG or the L1 monoclonal antibody Neuro4 complexed with F(ab′)2 fragments of anti-mouse IgG for 7 min. Cell extracts were subjected to SDS-PAGE and immunoblotting with anti-active MAPK antibody (p ERK2). Bottom, The same nitrocellulose filters were stripped and reblotted with an antibody recognizing ERK2 protein. ori, Origin;Rel, relative.