Ab155419

Western blotting to detect the eIF3d protein was performed using standard methods. Total protein was extracted from transfected cells with RIPA buffer, and the protein concentration was measured using a BCA Protein assay kit (Thermo Fisher Scientific, Inc.). The protein samples (30 µg) were subjected to SDS-PAGE (Bio-Rad, USA), transferred onto PVDF membranes and incubated overnight with primary antibodies at 4 °C. On the 2nd day, the membranes were incubated with secondary antibody at room temperature for 2 h. Immunoreactive bands were detected with an ECL western blotting system (Clarity Western ECL Substrate; Bio-Rad). The antibodies used in this study were as follows: rabbit anti-GAPDH (ab119716, Abcam), rabbit anti-eIF3d antibody (ab155419, Abcam), and goat anti rabbit IgG H&L (ab6721, Abcam).

After infection for 5 days, HCT116 cells were washed with ice-cold PBS and then lysed in 2× SDS sample buffer [100 mM Tris–HCl (pH 6.8), 10 mM EDTA, 4% (w/v) SDS, 10% (v/v) glycine] for 1 h at 4 °C. The lysates were clarified by centrifugation at 13 000× g for 30 min at 4 °C and the supernatants were employed for further analysis. The total protein concentration was estimated using BCA (bicinchoninic acid) protein assay kit. Protein samples (30 μg) were loaded and electrophoresed in a SDS–PAGE (10% gel) at 50 V for 3 h, and subsequently transferred to PVDF membranes (Millipore) at 300 mA for 1.5 h. After being blocked with TBST (Tris-buffered saline Tween-20) [20 mM Tris (pH7.6), 150 mM NaCl, 0.01% Tween-20] containing 5% (w/v) non-fat dried skimmed milk powder for 1 h at room temperature, membranes were probed with rabbit anti-EIF3D (abcam, #ab155419, dilution 1:1000), or mouse anti-GAPDH (Santa cruz, #Sc-32233, dilution 1:60 000) overnight at 4 °C. After washing by TBST, the membrane was incubated with HRP (horseradish peroxidase)-labelled anti-rabbit (Santa cruz, #Sc-2054, dilution 1:5000) or anti-mouse (Santa cruz, #Sc-2005, dilution 1:5000) secondary antibody at room temperature for 2 h. The membranes were analysed using super ECL detection reagent. Each experiment was repeated three times.