M per mammalian protein extract reagent

Protein lysates were obtained from HORCs using Mammalian Protein Extract Reagent M-PER supplemented with Halt Phosphatase Inhibitor Cocktail, Protease Inhibitor Cocktail and 5mM EDTA (All from Thermo Scientific, Loughborough, UK) for 20min on ice followed by centrifugation at 13,000rpm for 5min. Protein concentration of each lysate was determined using a bicinchoninic acid (BCA) protein assay (Thermo Scientific, Loughborough, UK). Equal amounts of protein were loaded onto 10% SDS-PAGE gels and proteins separated by electrophoresis. Proteins were transferred to PVDF membrane (Perkin Elmer Life Sciences, Cambridge, UK) using a semi-dry transfer blotter (Bio-Rad Laboratories, Hemel Hempstead, UK). Membranes were blocked with PBS-T (0.1% Tween-20 in PBS, 5% fat-reduced milk), hybridized with primary antibody followed by incubation with secondary antibody (GE Healthcare, Buckinghamshire, UK). Bands were visualised using chemiluminescent ECL Plus Western Blot Detection reagent (GE Healthcare, Buckinghamshire, UK) and net band intensity determined (1D 3.5 software, Eastman Kodak, Rochester, NY). Primary antibodies (Cell Signaling Technology, Danvers, MA, USA) against phospho- and total p38, phospho- and total JNK were used at 1:250, 1:1000, 1:500 and 1:500 respectively.

Protein extraction was performed by lysing explants in Mammalian Protein Extract Reagent M‐PER supplemented with Halt Phosphatase Inhibitor Cocktail, Protease Inhibitor Cocktail and 5 mM EDTA (All from Thermo Scientific, Loughborough, U.K.). Tissue was homogenized on ice for 20 minutes and then centrifuged at 13,000 rpm for 5 minutes to isolate the soluble cell extract. Protein concentration was determined using a bicinchoninic acid protein assay (Thermo Scientific, Loughborough, U.K.).
Ten micrograms of protein was loaded into 4%–12% precast gels and electro‐transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Scientific, Loughborough, U.K.). Membranes were blocked in 5% dried skimmed milk in PBS with 0.2% Tween20 (Sigma‐Aldrich, Poole, U.K.) for 1 hour and incubated overnight with primary antibody (Supporting Information Table 1) in blocking solution at 4°C. Horseradish peroxidase (HRP) conjugated secondary antibodies (1:10,000, Vector Laboratories Ltd., Peterborough, U.K.) were used for 2 hours before signal detection using ECL Prime (Amersham, GE Healthcare, U.K.) and an Alliance Western blot imaging system (UVItec Ltd., Cambridge, U.K.).