Figure 3

mSH3BGRL activates MAPK and AKT pathways in an FAK-dependent manner. (a) Lysates from stable cell lines were incubated with GST-Raf to enrich for GTP-bound active Ras. Eluates and total lysates were subsequently immunoblotted with various Ras/MAPK pathway-specific antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. (b) Lysates from (a) were immunoblotted with anti-p-FAK Y576/577 or FAK antibodies. (c) Lysates prepared as in (a) were incubated with GST-PBD (to enrich for GTP-bound active Cdc42 and Rac1). Elutes and total lysates were subsequently immunoblotted with antibodies against Cdc42 and Rac1. (d) CT-26 Vec, CT-26 SH3BGRL, CHO Vec and CHO SH3BGRL cells were transfected with FAK-specific siRNA, where indicated, for 48 h and lysed. Lysates were immunoblotted with the various antibodies listed. GAPDH was served as a loading control. The averaged relative protein expression level is quantified and shown under the immediate panels.