Ncor1

Protein samples were prepared in reduced and denatured forms (32 (link)) and resolved using SDS-PAGE. The proteins were transferred to a nitrocellulose membrane and then blocked with 5% nonfat milk in TBS-T (0.02 M Tris–HCl, 0.16 M NaCl, and 0.1% Tween-20, pH 7.4), at room temperature, for 1 h. The membranes were incubated overnight with primary antibodies PABPC4 (Bethyl, #A301-466A), NCoR1 (Affinity, #AF0270), PABPC1 (Thermo Fisher Scientific, #PA5-29883), FLAG (Sigma-Aldrich, #F1804), α-tubulin (Sigma-Aldrich, #T9026), OXPHOS (proteins of mitochondrial ETC) (Abcam, #ab110413), PPARD (Thermo Fisher Scientific, #PA1-823A), eIF4G (Cell Signaling Technology, #2498), puromycin (Merck, #MABE343), Vinculin (Cell Signaling Technology, #4650), Lamin A (Santa Cruz Biotechnology, #sc-71481), and β actin (Santa Cruz Biotechnology, #81178), Akt (Cell Signaling Technology, #9272), p-Akt^Thr308 (Cell Signaling Technology, #9275), ubiquitin (Abcam, #ab7254), and GST (Sigma-Aldrich, #G7781). The membrane was then washed with TBS-T and incubated with horseradish peroxidase–conjugated secondary antibody (1:10,000) in TBS-T solution containing 5% nonfat for 1 h. Membranes were washed with TBS-T and then added the peroxidase substrate SuperSignal West Plus (Thermo Fisher Scientific), and the band intensities were captured in the ImageQuant LAS500 (GE Healthcare).

Cells were grown in glass coverslips and treated as described in the legend of the figure. The media was removed, and the cells were washed twice with PBS. Cells were fixed with 4% paraformaldehyde in PBS, for 10 min, at room temperature and permeabilized with 0.1% of Triton X-1000 in PBS for 10 min, at room temperature. A blocking solution containing 3% BSA in PBS was added for 30 min, at room temperature, and the primary antibodies were diluted in 3% BSA, 0.1% Tween-20 in PBS (PABPC4, Bethyl, #A301-466A, 1:400 dilution; NCoR1, Affinity, #AF0270, 1:250 dilution). Samples were incubated overnight, at 4 °C, in a humidified chamber. The cells were washed three times with PBS and incubated with fluorescent-conjugated secondary antibody for 1 h, at room temperature, in the dark. The nucleus was stained with 1 μg/ml of Hoechst 33342 for 15 min, at room temperature, in the dark. The coverslips were then washed with PBS and mounted on glass slides using a hardening mounting medium (Dako).