SDS-PAGE and Western Blot Analysis of Protein Extracts

Cell extracts were prepared by lysing cells on ice for 15 minutes by the addition of lysis buffer containing 20 mmol/L Tris.HCl, 138 mmol/L NaCl, 2.7 mmol/L KCl, pH 8.0, supplemented with 5% glycerol, 1 mmol/L MgCl₂, 1 mmol/L CaCl₂, 1 mmol/L sodium‐o‐vanadate, 20 μmol/L leupeptin, 18 μmol/L pepstatin, 1% NP‐40, 5 mmol/L EDTA, and 20 mmol/L NaF. Cell debris and nuclei were removed by centrifugation at 10 000×g for 10 minutes at 4°C. Protein concentration was determined with the Bio‐Rad DC Protein Assay kit, according to the manufacturer's instruction. Extracts (40 μg) were subjected to SDS‐PAGE and were electrophoretically transferred to an Immobilon‐P membrane (Millipore), and the resultant membrane was incubated overnight with the corresponding first antibody at 4°C, with gentle agitation after blocking with 5% skimmed milk. The protein was then decorated with corresponding anti‐mouse (Alexa Fluor 680–conjugated) or anti‐rabbit (IRDye 800–conjugated) secondary antibodies. Signals were visualized with Odyssey Infrared Imaging System (LI‐COR Biosciences). Quantification of the signals was performed in NIH Image 1.62 software.