The MCF-7 cell line was maintained under standard conditions in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum. Cells were washed with ice cold phosphate buffered Saline and lysed in RIPA buffer (1% NP-40, 0.1% SDS, 0.5% Sodium deoxycholate, 50 mM Tris pH 7.5, 150 mM NaCl) supplemented with protease and phosphatase inhibitor cocktails (Sigma Aldrich) and protein concentration was quantitated by BCA protein assay (Invitrogen). Purified BSA (Applichem) was dissolved in RIPA buffer. Cell lysates and a BSA sample were serially diluted 1∶2 and run on SDS-PAGE using a standard protocol. Proteins were transferred to the PVDF (for ECL based detection) or Nitrocellulose (for LI-COR based proteins detection) membranes. Membranes were blocked with blocking solution (11500694001, Roche) for BSA detection or 5% skimmed milk for rest of the membranes. For Western blotting ERK (M-5670, Sigma Aldrich), mTOR (2972, Cell Signaling Technology), RSK1 (sc-231, Santa Cruz) and BSA (sc-50528, Santa Cruz) antibodies were used. Anti-rabbit HRP-conjugated (Cell Signaling Technology) or anti-Rabbit IR 800 (LI-COR) secondary antibodies were used for ECL or LI-COR protein detection systems, respectively. Signal was detected by standard X-ray films (Fuji), CCD camera (Advanced Molecular Vision) or LI-COR scanner.
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