Secondary antibodies conjugated to horseradish peroxidase

Total cell lysates and immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted by a semi-dry transfer in an electric field onto polyvinylidene difluoride membranes (PVDF; Millipore). After transfer, the membranes were blocked in 5% low fat dry milk in TBST (10 mM TRIS-HCl, 100 nM NaCl and 0.05% Tween 20; pH 7.4) for 1 h at room temperature and incubated overnight at 4°C with a primary antibody in 1% low fat dry milk in TBST. The following primary antibodies were used: anti-TFII-I (V-18; Santa Cruz Biotechnology), anti-GST (GE Healthcare), anti-Mdm2 (Ab-1; Merck-Millipore), anti-Tubulin alpha (Exbio), anti-PCNA, (PC-10, kindly provided by Borivoj Vojtesek, Masaryk Memorial Cancer Institute, Brno, Czech Republic), anti-β-galactosidase (BG-02; Santa Cruz Biotechnology), anti-Myc (9E10, Santa Cruz Biotechnology), anti-Flag (M2; Sigma-Aldrich) and anti-GFP (Roche), using dilutions recommended by the manufacturers. After three washes in 1% milk in TBS, membranes were incubated one hour at room temperature with secondary antibodies conjugated to horseradish peroxidase (GE Healthcare). Enhanced chemiluminescence kit (ECL; GE Healthcare) and blue light sensitive medical X-ray film (Agfa) or G:BOX Chemi imaging system (Syngene) were used to visualize proteins of interest.

Immunoblotting was performed as described previously^{50 (link)}. Total protein extracts were obtained from homogenised tissues and cultured cells, and lysed with lysis buffer. To fractionate the cell lysate into cytoplasmic and nuclear fractions, the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Fisher Scientific) was used. Protein concentrations of all samples were measured using a Bradford protein assay (Bio-Rad, CA, USA). Tissue and cell lysates were separated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (PVDF). After membranes were cut for blocking with an appropriate blocking reagent, the membranes were blocked with tris-buffered saline containing 0.1% Tween 20, 5% non-fat dry milk (for proteins except NFATc1, Phospho-SAPK (Stress-activated protein kinase)/JNK and SAPK/JNK), 5% bovine serum albumin (for NFATc1), or 2.5% non-fat dry milk and 2.5% bovine serum albumin (for Phospho-SAPK/JNK and SAPK/JNK). Membranes were then incubated overnight with appropriate primary antibodies (Supplementary Table 3). Secondary antibodies conjugated to horseradish peroxidase (GE Healthcare, Buckinghamshire, UK) were used for detection with enhanced chemiluminescence (PerkinElmer, MA, USA). The Western blotting data was found using ImageQuant TL Image Analysis Software (Cytiva, MA, USA).