Figure 4. NgBR over-expression promotes Ras membrane accumulation and EGF-stimulated Ras activation.

(A) Over-expression of NgBR increases the NgBR protein levels in the fraction of biotinylated cell surface proteins. NIH-3T3 cell surface proteins were biotinylated under non-permeabilized conditions and isolated using streptavidin agarose resin from the Pierce Cell Surface Protein Isolation Kit as described in Figure 1B. Proteins were determined by Western blot analysis using antibodies that detect endogenous proteins. (B) Over-expression of NgBR in NIH-3T3 cells promotes H-Ras and K-Ras membrane localization. The plasma membrane proteins were isolated by the ultracentrifugation method. The protein levels were determined by Western blotting using antibodies that detect endogenous proteins. (C) Over-expression of NgBR in NIH-3T3 cells increases the EGF-induced H-Ras, K-Ras and pan-Ras activation. The activated Ras proteins were isolated using GST-RBD beads and protein levels were determined by Western blotting. (D) Over-expression of NgBR in NIH-3T3 cells increases the EGF induced phosphorylation of Akt and ERK. Phosphorylation of Akt and ERK was determined by Western blotting using phosphorylation specific antibodies. Data are validated in 3 independent experiments.