Protran 0.2 mm nitrocellulose

Cells were rinsed in ice-cold PBS then lyzed in NP40 lysis buffer (1% NP40, 50 mM Tris-HCl pH 8.0, 150 mM NaCl) plus protease inhibitor cocktail (Pierce), 5 mM sodium fluoride, 2 mM sodium orthovanadate and 0.2 mM PMSF incubating on ice for 15 min. Lysates were cleared by centrifugation at 20,000 g for 15 min at 4°C then protein concentrations determined using Coomassie Protein Assay Kit (Thermo Scientific, UK). For each sample, 30 μg of total protein were separated on 7% Bis-Tris polyacrylamide gels in SDS running buffer then blotted onto Protran 0.2 mM Nitrocellulose (GE Healthcare, UK). Membranes were probed with 1:1000 dilution of the appropriate primary antibody (mouse anti-gp130; Santa Cruz sc376280), rabbit anti-Clathrin (Biolegend, 813901), STAT3 (Cell Signaling Technologies, #9139), P-STAT3 (Y705, Biolegend, #651006) P-STAT1 (Y701, Cell Signaling Technologies, #8009) or 1:5000 dilution mouse anti-GAPDH (Cell Signaling Technologies, #2118), then 1:5000 dilution of donkey anti-rabbit-HRP (Stratech, 711-035-152-JIR) or donkey anti-mouse-HRP (Stratech, 715-035-150-JIR) as the secondary antibody. Immobilon Western Chemiluminescent HRP substrate (Millipore, UK) was used for visualization.

Cells were rinsed in ice-cold PBS then lyzed in RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (ROCHE), 5 mM sodium fluoride, 2 mM sodium orthovanadate and 0.2 mM PMSF incubating on ice for 15 min. Lysates were cleared by centrifugation at 20,000 g for 15 min at 4°C then protein concentrations determined using Coomassie Protein Assay Kit (Thermo Scientific, UK). For each sample, 30 μg of total protein were separated on 4%–12% Bis-Tris polyacrylamide gels (NuPAGE, Invitroge) in MES SDS running buffer then blotted onto Protran 0.2 mM Nitrocellulose (GE Healthcare, UK). Membranes were probed with 1:1000 dilution of the appropriate primary antibody anti-total-STAT3 (Cell Signaling, #9139S), anti-total-RPB1 (Cell Signaling, #14958), anti-pSer2-RPB1 (CellSignaling, #13499), anti-pSer5-RPB1 (CellSignaling, #13523) and anti-GAPDH (Cell Signaling, #2118S). 1:5000 dilution of donkey anti-rabbit-HRP (Stratech, 711-035-152-JIR) or donkey anti-mouse-HRP (Stratech, 715-035-150-JIR) as the secondary antibody. Immobilon Western Chemiluminescent HRP substrate (Millipore, UK) was used for visualization.