3 3 5 5 tetramethylbenzidine tmb liquid substrate

After SDS-PAGE and 2D electrophoresis, gels were placed on polyvinylidene difluoride (PVDF) membranes and electrically transferred. The total amount of protein loaded and transferred was controlled before immunodetection by staining the membrane with 0.1% (w/v) Coomassie Brilliant Blue R-250 in isopropanol. Membranes were destained for 15 min in acetic acid/ethanol/water (1:5:4), washed with water and finally airdried. The membranes were then blocked by incubation with 5% (w/v) non-fat milk in 20 mM Tris, 150 mM NaCl (pH 7.6 )) for 1 h at 20°C. The membranes were washed three times with TBST, and then incubated for 16 h at 4°C with polyclonal antibodies to either phosphotyrosine, phosphoserine, a phosphothreonine, PKA, PKC, phospho-(Ser/ Thr) PKA substrates or phospho-(Ser) PKC substrates diluted 1:1000 in 5% w/v bovine serum albumin in TBST (BSA-TBST). Membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000 in 3% w/v BSA-TBST) for 1 h at 20°C. Reacted proteins were detected either with 3,3′,5,5′ tetramethylbenzidine (TMB) liquid substrate (Sigma-Aldrich) or with ECL-plus (GE Healthcare). The relative intensity of each band was measured using ImageJ (NIH, Bethesda, MD, USA). Total protein staining was used as a loading control (Supplementary Fig. 1, see section on Supplementary Data given at the end of this article).