Chemidoc chemoluminescence detection system

Confluent cells were lysed in 100 mM Tris pH 7.5,150 mM NaCl, 0.5% NP40, 0.5% triton-X100, 10% glycerol,1X protease inhibitor cocktail (Roche) and 1X phosphatase inhibitor (Phosphostop, Roche) for 20 min at 4°C. Insoluble debris were centrifuged for 15 min at 13000 g and supernatants were recovered. Protein concentration was quantified by Bradford assay (BioRad), SDS PAGE and electrotransfer were performed on 4–12% Bis-Tris gel (Novex) using mini gel tank and iBlot transfer systems (Invitrogen). Non-specific sites were blocked with 5% non-fat dry milk in PBS 0.1% Tween 20. Primary antibodies were diluted (1/1000) in PBS 0.1% Tween 20 and incubated overnight at 4°C. After three washes in PBS 0.1% Tween 20, secondary HRP antibodies diluted in PBS 0.1% Tween 20 (1/10000) were incubated for 1 hr and washed 3 times with PBS 0.1% Tween 20. Immunocomplexes of interest were detected using Supersignal west femto maximum sensitivity substrate (ThermoFisher) and visualized with ChemiDoc chemoluminescence detection system (Biorad). Quantification of Western blots by densitometry was performed using the Gel analyzer plug in from Image J. GADPH was used as a loading control to normalize the quantification.

Confluent cells were lysed in 100 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP40, 0.5% triton-X100, 10% glycerol,1X protease inhibitor cocktail (Roche) and 1 X phosphatase inhibitor (Roche) for 20 min at 4 °C. After 15 min centrifugation at 13,000 g, solubilized proteins were recovered in the supernatant. Protein concentration was measured using Bradford assay (Bio-Rad). For SDS PAGE, 50 µg protein extracts were loaded in 4–12% Bis-Tris gel (Invitrogen) or poly-acrylamide gels and proteins were transferred overnight at 4 °C on a nitrocellulose membrane using a liquid transfer system (Bio-Rad). Non-specific sites were blocked with 5% non-fat dry milk in PBS 0.1% Tween 20. Primary antibodies were diluted (1/1000 to 1/500) in PBS 0.1% Tween 20 and incubated overnight at 4 °C. After three washes in PBS 0.1% Tween 20, secondary HRP antibodies diluted in PBS 0.1% Tween 20 (1/10,000) were incubated for 1 hr and washed three times with PBS 0.1% Tween 20. Immunocomplexes of interest were detected using Supersignal west femto maximum sensitivity substrate (Thermo Fisher Scientific) and visualized with ChemiDoc chemoluminescence detection system (Bio-Rad). Quantification of Western blots by densitometry was performed using the Gel analyzer plug in from Image J.