Dc detergent compatible assay

Total cell lysates were obtained and separated by SDS-PAGE. Briefly, cells were solubilized with lysis buffer containing 50mM HEPES, 150mM NaCl, 10mM EDTA, 10mM Na₄P₂O₇, 2mM sodium orthovanadate, 50mM NaF, 1mM phenylmethylsulfonyl fluoride, 10μg/ml aprotinin, 10μg/ml leupeptin, pH 7.4, and 1% (v/v) Triton X-100. Lysates were clarified by centrifugation at 12,000g for 20 minutes at 4°C. The protein concentrations in the cell lysates were measured using a Bio-Rad DC (detergent compatible) assay. Immunoprecipitation and Western blot analysis have been performed as previously described [37 (link)].

Total cell lysates were obtained and separated by SDS-PAGE. Briefly, cells were solubilized for 20 min at 4 C with lysis buffer containing 50 mM HEPES, 150 mM NaCl, 10 mM EDTA, 10 mM Na₄P₂O₇, 2 mM sodium orthovanadate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, pH 7.4, and 1% (v/v) Triton X-100. Lysates were clarified by centrifugation at 12,000g for 20 min at 4 C. The protein concentrations in the cell lysates were measured using a Bio-Rad DC (detergent compatible) assay. As already described [25 (link),30 (link)], the same amount (50/80 μg of protein/lane) of proteins were denatured by boiling in Laemmli sample buffer containing 10% 2-mercaptoethanol. Proteins were separated by SDS-polyacrylamide gel electrophoresis and blotted on Immobilon-P membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 1 h in TBS tween (10 mM Tris-HCl, pH 7.4, and 140 mM NaCl) containing 3% (w/v) bovine serum albumin and then incubated with the indicated antibodies.
Detection of blotted proteins was performed by ECL according to the manufacturer's instruction. Densitometric analysis was performed using Image Lab software 3.0 (Bio-Rad, Hercules, CA, USA). To evaluate insulin response, cells were serum starved for 24 h in media containing 0.25% BSA, with or without 1 nM BPA and then stimulated with 100 nM of insulin for 10 min.