Chemidoc xrs densitometer system

After harvesting, the cells were lysed in 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 1% nonidet P-40, protease inhibitor cocktail, 1 mM phenylmethane sulfonyl fluoride, 25 mM sodium fluoride, and 1 mM sodium orthovanadate. Protein concentrations were determined using BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Subsequently, cellular extracts (45~60 μg) were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Non-specific binding was blocked with 5% skim milk in Tris-buffered saline with Tween 20 for 30 min at room temperature. The membranes were then incubated with the primary antibody (1 : 1,000) overnight at 4°C and continually with the secondary antibody (1 : 5,000) at room temperature for 30 min. After incubation, the membranes were washed three times for 10 min each. Proteins were visualized using ECL reagent, and the band intensity was measured using a Chemidoc XRS densitometer system and quantified by Quantity One software (Bio-Rad).

Following transfection, whole cell lysates were prepared and protein concentration was determined using BCA Protein Assay Reagents. Cellular extracts (20 μg) were separated on 10% SDS-PAGE at 100 V and transferred onto 0.45 μm PVDF membrane. Nonspecific binding was blocked with 5% nonfat milk in TBS-T for 1 h at room temperature. Primary antibody was used at a 1:1000 dilution. Secondary antibody was used in a 1:5000 dilution. The incubation of primary antibodies was done at 4°C for overnight incubation. Secondary antibodies were done at 4°C for 2 h. Proteins were visualized by an ECL method and the band intensity was analyzed by Chemidoc XRS densitometer system and quantified by Quantity One software (Bio-Rad).