Cells were lysed in sample buffer containing β-mercaptoethanol at room temperature. Lysates were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and blotted using nitrocellulose membrane. Blots were blocked and probed using 3% milk/PBS–0.2% Tween 20 or 5% bovine serum albumin (BSA)/tris-buffered saline (TBS)–0.1% Tween 20 and quantified using ECL Plus Western blot detection system (GE Healthcare). For IP experiments, GFP, CAR-GFP A549, or wild-type A549 expressing FLAG-tagged constructs were lysed in IP lysis buffer [50 mM tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% NP-40, and protease inhibitor cocktail]. Lysates were incubated with 5 μg of anti-GFP or anti-FLAG antibody prebound to A/G agarose beads overnight before washing the beads with 1 ml of IP lysis buffer three times. Immunocomplexes were separated using SDS-PAGE and immunoblotted for specified proteins. Where relevant for quantification purposes, phospho-proteins levels were normalized to levels of the same total protein before comparing between conditions.

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