Sds 4x sample buffer

The cells were harvested and lysed in an immunoprecipitation lysis buffer (50 mM NaCl, 0.5% Nonidet P-40, and 10 mM Tris-HCl, pH 8.0) containing EDTA-free protease inhibitor cocktail tablets (Complete Mini, EDTA-free) on ice for 30 min. Further, equal concentrations of proteins were denatured with sodium dodecyl sulfate (SDS) 4X sample buffer (Bio-Rad, Hercules, CA, USA) containing 1% 2-ME (2-mercaptoethanol) and heated at 95 °C for 10 min. These samples were separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. After incubation with 5% nonfat milk in TBST (Tris-buffered saline with 0.1% Tween 20) for 1 h, membranes were incubated with primary antibodies overnight at 4 °C (Table S1). The membranes were washed three times with TBST for 10 min, followed by incubation with secondary antibodies for 1 h at room temperature. Blots were washed three times for 10 min with TBST and developed using the Azure Biosystem 400, and the band intensity was analyzed using the Image Studio Lite software (LiCor 5.2.5, Lincoln, NE, USA).

Transfected cells were harvested and lysed using the modified radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% sodium dodecyl sulfate [SDS], 0.5% Triton X-100, and 0.05% sodium deoxycholate [SigmaAldrich, D6750]) supplemented with EDTA-free protease inhibitor cocktail tablets (Complete Mini, EDTA-free; Roche Diagnostics, 11,836,170,001) on ice for 30 min. Further, an equal amount of proteins was denatured with SDS 4X sample buffer (Bio-Rad, 161–0747) containing 1% 2-mercaptoethanol and heated at 95°C for 10 min. These protein samples were separated by using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane. After incubation with 5% nonfat milk in TBST (Tris-buffered saline with 0.1% Tween 20 [Bio-Rad Laboratories, 170–6531]) for 1 h, membranes were incubated with various primary antibodies overnight at 4°C. Membranes were washed 3 times with TBST for 10 min followed by probing with secondary antibody for 1 h at room temperature. Blots were washed 3 times for 10 min with TBST and developed by using the Azure Biosystem C600 and band intensity was analyzed by Image Studio Lite (LiCor 5.2.5) according to the manufacturers manual.