Unless indicated otherwise, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in ice-cold buffer A containing 0.5% NP-40 and 0.1% Brij-35, as described (45). To maintain the detergent-sensitive mTOR-raptor interaction, cells were lysed in ice-cold buffer A containing 0.3% CHAPS. Lysates were spun at 13,200 rpm for 5 min at 4°C, and the post-nuclear supernatants were collected and incubated on ice (15 min). The Bradford assay was used to normalize protein levels for immunoprecipitation and immunoblot analysis. For immunoprecipitation, whole-cell lysates were incubated with antibodies for 2 hours at 4°C, followed by incubation with protein G- or A-Sepharose beads for 1 hour. Sepharose beads were washed three times in lysis buffer and resuspended in 1× sample buffer. Samples were resolved on SDS-PAGE and transferred to PVDF membranes in Towbin transfer buffer containing 0.02% SDS, as described (45). Immunoblotting was performed by blocking PVDF membranes in tris-buffered saline (TBS; pH 7.5) with 0.1% Tween 20 (TBST) containing 3% nonfat dry milk, as described (45), and incubating the membranes in TBST with 2% bovine serum albumin (BSA) containing primary antibodies or secondary HRP-conjugated antibodies. Blots were developed by ECL and detected digitally with a ChemiDoc-It System (UVP).

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