Western Blotting of eIF6 in HCT116 Cells

HCT116 cells (human colorectal carcinoma line) (ATCC) were cultured as described previously (37 (link),57 (link)). HCT116 cells were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS), and 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco). Cells are routinely ensured to be negative for mycoplasma contamination using the mycoplasma detection kit (ATCC). For western blotting, the cells were washed twice in PBS and lysed in mammalian cell lysis buffer (MCLB) (50 mM Tris–Cl pH 8.0, 5 mM EDTA, 0.5% Igepal, 150 mM NaCl) that was supplemented with the following inhibitors just before lysis: 1 mM phenylmethylsufonyl fluoride (PMSF), 1 mM sodium fluoride, 10 mM β-glycerophosphate, 1 mM sodium vanadate, 2 mM DTT, 1× protease inhibitor cocktail (Sigma–Aldrich), 1× phosphatase inhibitor cocktail (Santa Cruz Biotechnology). Lysates were rocked for 15 min at 4°C followed by centrifugation at 14 000 rpm for 10 min at 4°C. Blots were probed with anti-eIF6 antibody (Santa Cruz Biotechnology, sc-390441) (1:1000, overnight) or anti-βTubulin antibody (Cell Signaling, 2128) (1:1000, overnight) diluted in TBS-T buffer.