Clarity western ecl

Total proteins were extracted from MSB1 cells 48h post-transfection using radio immunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors. The concentration of proteins was determined by the BCA assay (BCA Protein Assay Kit, Beyotime, Shanghai, China), and 20µg protein per sample was denatured in loading buffer by boiling for 3~5 min, and separated by 10% SDS-PAGE. The resulting bands were electro-transferred to polyvinylidene di uoride (PVDF) membrane at 100 mA over 1.5h. After blocking with 4% BSA for 1h, the membranes were incubated overnight with primary antibodies against RORA (1:1000, Abcam, ab60134) and β-actin (1:1,000, Abcam, ab8226), followed by HRP-conjugated anti-rabbit IgG (1:1000) and anti-mouse IgG (1:1000) (Bayotime) respectively. The positive proteins bands were detected using a chemiluminescence system (Bio-Rad Clarity Western ECL; Bio-Rad Laboratories Inc.), and the grayscale values were quanti ed using ImageJ. The density of the RORA bands was standardized to that of β-actin.

Total proteins were extracted from MSB1 cells 48h post-transfection using radio immunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors. The concentration of proteins was determined by the BCA assay (BCA Protein Assay Kit, Beyotime, Shanghai, China), and 20µg protein per sample was denatured in loading buffer by boiling for 3~5 min, and separated by 10% SDS-PAGE. The resulting bands were electro-transferred to polyvinylidene di uoride (PVDF) membrane at 100 mA over 1.5h. After blocking with 4% BSA for 1h, the membranes were incubated overnight with primary antibodies against RORA (1:1000, Abcam, ab60134) and β-actin (1:1,000, Abcam, ab8226), followed by HRP-conjugated anti-rabbit IgG (1:1000) and anti-mouse IgG (1:1000) (Bayotime) respectively. The positive proteins bands were detected using a chemiluminescence system (Bio-Rad Clarity Western ECL; Bio-Rad Laboratories Inc.), and the grayscale values were quanti ed using ImageJ. The density of the RORA bands was standardized to that of β-actin.

For extracting total protein, cells were resuspended in lysis buffer containing 150mmol/L NaCl, 1%SDS, and 50 mmol/L Tris-Cl pH 8, 2ug/mL aprotinin 2ug/mL leupeptin, 40mg/mL of phenylmethylsulfonyl uoride (PMSF), 2mmol/L dithiothreitol (DTT). Palate centrifuged at 12000 rpm for 15 min and supernatant were then quickly frozen at -80 C until use.
The protein concentration was distincted by the Bradford assay (Biorad, Hercules, and CA). Thirty micrograms of cell extracted protein from B16F10 were loaded onto a 10% SDS-polyacrylamide gel subjected to electrophoresis and electro-transferred onto a nitrocellulose membrane. The immunoblot was incubated 1 h at room temperature with 5% nonfat milk, and then the membrane was then probed with the primary antibody overnight at 4 C. The next day, Blots were washed twice with Tris buffered saline/Tween 20(TBST) and the secondary appropriate antibody at room temperature was added for 1 h. Blots were again washed with TBST before development. The visualization blot of the signal was obtained using ECL (Bio-Rad Clarity Western ECL). Band densitometry was measured by Image J software, Protein Simple and semi quantitative data were normalized for GADPH and Tubulin.