X ray film

Cells were washed with ice-cold PBS and then directly lysed in cold RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). Equal amounts of protein (20 μg) were boiled in 2x Laemmli Sample Buffer (Bio-Rad Laboratories) and separated by SDS-PAGE (4–15%, Tris-HCl) using the Bio-Rad Criterion system. Separated proteins were then transferred onto nitrocellulose membranes (Bio-Rad Laboratories) and blocked with 5% nonfat milk in PBS-0.05% Tween-20 for at 1 hour at room temperature. Anti-phospho-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-SK1 and anti-GAPDH were from Cell Signaling Technology (Danvers, MA, USA). Anti-COX2 was obtained from BD Biosciences (Franklin Lakes, NJ, USA). Horseradish peroxidase-labeled secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). Primary antibodies diluted 1:1000 or 1:5000 for GAPDH were then added to membranes and incubated at 4°C overnight. Membranes were washed 3 times with PBS-0.05% Tween-20 and then incubated with diluted 1:5000 horseradish peroxidase conjugated secondary antibodies for 1 hour at room temperature. Membranes were washed 3 times with PBS-0.05% Tween-20 and then incubated with Pierce ECL Substrate and exposed to X-ray films (Thermo Fisher Scientific) that were then processed and scanned.