FIG. 7.

Expression of GIPC(LG>AE) inhibits NGF-induced neurite outgrowth and MAP kinase activation. (A) Bar graph showing the percentages of cells that have differentiated cells expressing different GIPC constructs. In nontransfected cells that were not stimulated with NGF (−), only 4% of the cells differentiated and grew out at least one neurite. After NGF treatment (+), 50% of the nontransfected cells, 41% of the mock-transfected cells (empty vector), and 37% of the GIPC-transfected cells differentiated, whereas in the cells transfected with the GIPC(LG>AE) mutant, only 27% of the cells differentiated. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG>AE), incubated with NGF (50 ng/ml) for 16 h, fixed, permeabilized, and incubated with anti-FLAG to label transfected cells. Cells were scored for differentiation (neurite outgrowth) as described in Materials and Methods. Average differentiation rates (±standard errors) are for six experiments (≥50 cells/experiment). **, P ≤ 0.01. (B) Overexpression of the FLAG-tagged GIPC(LG>AE) mutant strongly inhibits NGF-induced Erk1/2 activation. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG>AE), serum starved, treated with NGF (100 ng/ml) for 5 min, and lysed for immunoblotting with anti-pErk1/2 antibody. Per lane, 20 μg protein was loaded; the levels of total Erk1/2 (bottom) are the same in all samples. (C) Quantification of the pErk1/2 levels from panel B, shown as normalized percentages of the mock control.