Hrp conjugated secondary antibody

SDS‐PAGE and subsequent western blotting analyses were performed as previously described.29 Cell lysates were prepared by solubilizing cells in radio‐immunoprecipitation assay (RIPA) buffer (50 mM Tris‐HCl [pH 7.5] containing 150 mM NaCl, 1% NP‐40, and 0.1% sodium deoxycholate) supplemented with protease‐inhibitor and phosphatase‐inhibitor cocktails (Sigma). Samples were then mixed at a 1:4 ration with 5 × sample buffer (300 mM Tris‐HCl [pH 6.8] containing 10% 2‐mercaptoethanol, 10% SDS, 50% glycerol and 0.05% Coomassie Brilliant Blue) and boiled for 5 min. The resulting samples (20 μg protein/lane) were subsequently separated by SDS‐PAGE using pre‐cast 5%–20% Tris‐glycine gradient gels (Atto Corporation, Tokyo, Japan) and transferred to PVDF membranes (Wako Pure Chemical). Membranes were blocked by incubation with TBS containing 0.1% Tween 20 (TBS‐T) and 5% skim milk (Megmilk Snow Bland, Tokyo, Japan) or BSA (Sigma) for 1 h, probed with the appropriate primary antibodies diluted with Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan) at 4°C, washed extensively with TBS‐T, and incubated with HRP‐conjugated secondary antibodies (1:5000 dilution) (GE Healthcare, Little Chalfont, UK). Finally, the membranes were washed, developed by incubation with ECL detection reagents (GE Healthcare) and exposed to Hyperfilm ECL (GE Healthcare).

Cells were treated with the various drugs and then lysed as described previously.18, 21 To prevent post‐lysis changes in phosphorylation, 50 mM NaF and 1.68 mM Na₃VO₄ were added. After centrifugation (11700 g for 10 min), the protein concentrations of lysates were evaluated by the Bradford method, and sample buffer containing 5% 2‐mercaptoethanol was added. After boiling for 5 min, 20‐μg samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and blocked with 5% skim milk in TBS‐tween buffer (50 mM Tris‐HCL [pH 7.4], 150 mM NaCl and 0.05% Tween 20). Immunoblotting was performed using primary antibodies appropriately diluted in TBS‐tween buffer containing 5% BSA and 0.05% sodium azide (Table S1). HRP conjugated secondary antibodies (GE Healthcare, Little Chalfont, UK) were added and activated with Immobilon Western Substrate (Millipore, Billerica, MA, USA). Images were obtained using a LAS 4000mini bio‐imager (Fujifilm, Tokyo, Japan) and analyzed with MultiGauge software (Fujifilm).