Fig. 5. 9β1 integrin-dependent signaling induced by mNGF. (A) 9SW480 cells were allowed to adhere to immobilized mNGF (20 µg/ml) or BSA for 30 minutes in the absence or presence of 9β1 integrin inhibitors, Y9A2 (10 µg/ml) or VLO5 (10 µg/ml). Cell lysates were obtained and equal amounts of protein were separated under reducing conditions by 10% SDS-PAGE. The proteins from the gel were electro-transferred onto a PVDF membrane and incubated with primary anti-phospho-Erk1/2 (Thr202/Tyr204) and anti-Erk1/2 polyclonal antibodies. The bands were visualized using chemiluminescent western detection kit. The numbers above the bands represent the average number of pixels, reflecting intensity of the bands in the presented scans, digitalized using Un-Scan-It gel software. (B) Paxillin phosphorylation in 9SW480 cells induced following binding to immobilized mNGF. Anti-phospho-paxillin (Tyr31) and anti-paxillin polyclonal antibodies were used. (C) Effect of mNGF on phosphorylation of Erk1/2 in GD10 cells transfected with β1 subunit or hybrid β1/β3 subunit and co-transfected with 9 integrin subunit. Cells were cultured on the plate and stimulated with or without mNGF (50 ng/ml) for 1 hour. Cells were lysed and detection of phosphor-Erk1/2 and total Erk1/2 was performed as described above. All signaling experiments were repeated at least three times.