Erk2 antibody

Total cell lysates were separated on 10% denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. Membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) plus 0.1% Tween 20 (PBST) buffer for 1 h at room temperature, were washed with PBST three times and then incubated with primary antibody overnight at 4 °C. ERK2 antibody was from Santa Cruz Biotechnology (Cat. No. sc-154) and was used at a dilution of 1:5000. The antibodies against mitofusin 2 and OPA1 were from Santa Cruz Biotechnology (Cat. No. sc-100560) and BD Transduction Laboratories (Cat. No. 612606) respectively, and used at dilutions of 1:250 and 1:1000. NCLX antibody was kindly provided by Prof. Israel Sekler and was used at 1:1000–1:2000 dilution. The membranes were then washed with PBST again and incubated with 1:2500 dilutions of peroxidase-linked anti-rabbit IgG from Santa Cruz Biotechnology (Cat. No. sc-2004) or anti mouse IgG from BD Bioscience (Cat. No. 554002) for 1 h at room temperature. After washing with PBST, the bands were detected by an enhanced chemiluminescence plus western blotting detection system (Amersham Biosciences). Blots were analysed by UN Scan software.

The Met polyclonal antibody, kindly provided by Dr. Morag Park (McGill University, Montreal, QC, Canada), was raised against an epitope in the C-terminal region of human Met, distinct from those altered in the variants (Additional file 1) [8 (link),21 (link)]. The Phospho-Tyr (p-Tyr100), phospho-Akt (Ser473), and phospho-Erk1/2 (p44/42MAPK, Thr202/Tyr204) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The pan-Shc and phospho-Tyr Shc (Tyr239/240) antibodies, that recognize the p66, p52, and p46 isoforms of ShcA, and the Erk2 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The α-tubulin and β-actin antibodies were from Sigma-Aldrich Canada Ltd (Oakville, ON, Canada). The Grb2 and E-cadherin antibodies were purchased from BD Transduction Labs (Lexington, KY, USA). The MEK1/2 and PI3K inhibitors, U0126 and LY294002, were purchased from Cell Signaling Technology, while the MEK1/2 inhibitors AZD6244 and PD184352 were obtained from Selleck Chemicals (Houston, TX, USA).