Cell lysates were collected in 1% NP-40 buffer and quantified by BCA protein assay (Thermo Scientific). Proteins were separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad) by semi-dry transfer at 25V for 40 minutes. All membranes were blocked in 5% milk in Tris-Buffered Saline (TBST) and probed overnight with indicated antibodies, which are diluted in the blocking buffer solution at 4°C. Primary antibodies included: mouse Flag (Sigma), rabbit V5 (Bethyl Laboratories), rabbit HA (Covance), mouse panAkt (Cell Signaling). HRP-conjugated secondary antibodies were incubated on membranes in 5% milk in TBST and bands were developed with ECL reagent (Thermo Scientific) and imaged using a Fuji LAS-4000 imager.
For immunoprecipitation, cells were harvested and then lysed in 1% NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). After pre-clearing with protein A/G agarose beads for 1 h at 4°C, whole cell lysates were used for immunoprecipitation with the indicated antibodies. 30 µl of Flag M2 beads (Sigma) was added to 1 ml of cell lysates and incubated at 4°C for 4 h. Immunoprecipitates were extensively washed 5 times with lysis buffer and eluted with SDS loading buffer by boiling for 5 min.
For immunoprecipitation, cells were harvested and then lysed in 1% NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). After pre-clearing with protein A/G agarose beads for 1 h at 4°C, whole cell lysates were used for immunoprecipitation with the indicated antibodies. 30 µl of Flag M2 beads (Sigma) was added to 1 ml of cell lysates and incubated at 4°C for 4 h. Immunoprecipitates were extensively washed 5 times with lysis buffer and eluted with SDS loading buffer by boiling for 5 min.