Phospho erk1 2

Cell lysates were separated by SDS/PAGE (8, 10 or 12% polyacrylamide gel), electrotransfered to PVDF membrane (Millipore,) and blocked for 1 h (at room temperature) in 5% non-fat powdered milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (BioShop, Burlington, Canada). Membranes were incubated with primary antibody overnight at 4 °C. After incubation with secondary antibody for 1 h, room temperature, chemiluminescence was detected using Immobilon Western HRP substrate (Millipore) with ChemiDoc system (BioRad). The following antibodies and dilutions were used: rabbit anti-MCPIP1 (1:1000, GeneTex), HIF1α (1:1000), HIF2α (1:1000), VHL (1:500), Akt (1:1000), Phospho-Akt (1:2000), SAPK/JNK (1:500), Phospho-SAPK/JNK (1:1000), p38 (1:1000), Phospho-p38 (1:1000), ERK1/2 (1:1000), Phospho-ERK1/2 (1:1000). All mentioned antibodies were from Cell Signaling Technology. Tubulin (1:4000, Calbiochem; Merck Millipore, Billerica, MA, USA) or in case tissue samples, GAPDH (1:30,000 Sigma-Aldrich) antibodies were used as a loading control. The following secondary antibodies were used: peroxidase-conjugated anti-rabbit IgG (1:30,000; Sigma) and peroxidase-conjugated anti-mouse IgG (1:10,000, Sigma).

For cell lysis, harvested samples were incubated on ice in whole-cell-extract lysis buffer for 30 min. Lysates were then centrifuged at 12,000 rpm, 4℃, for 10 min, after which protein concentration was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). For Western blot analysis, 15–100 μg of lysates (depending on the targeted proteins) were boiled for 5 min with sample buffer before being separated on sodium dodecyl sulfate–polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and blocked with 5% nonfat milk/TBST buffer. Primary antibodies used were as follows: HER2, beta-actin (Merck KGaA), ERα, ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology, Beverly, MA, USA), p62, phospho-ERK1/2 (GeneTex, Irvine, CA, USA), and LC3 (MBL international, Woburn, MA, USA). Anti-PARP antibody can also recognize cleaved PARP proteins. Anti-rabbit and anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). ImageJ software (National Institutes of Health, USA) was used to determine the intensity of each blot relative to the control after adjustment based on the intensity of beta-actin.