Anti rabbit immunoglobulin

Total protein was extracted from cell lines using lysis buffer (Thermo Fisher Scientific), containing a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China), and quantified using the Bradford method. Next, 40 µg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (8%), and the separated proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in PBS, the membranes were incubated overnight at 4°C with primary antibodies against: TRAF4 (H2818, 1:100, Santa Cruz Biotechnology), Eg5 (1:1000), Smurf2 (F0641, 1:100, Santa Cruz Biotechnology), HA (TA180128S, 1:1000, Origene), α-tubulin (ab52866, 1:1000, Abcam), Caspase-3 (M005851F, 1:1000, Abmart), Bcl-2 (T40056F, 1:1000, Abmart), Bax (T40051F, 1:1000, Abmart), Ki67 (550609, 1:1000, BD Pharmingen), GAPDH (AF0006, 1:2000, Beyotime) and β-actin (1:2000, Beyotime). Next, the membranes were incubated with secondary HRP-conjugated antibody, anti-mouse immunoglobulin G (IgG), or anti-rabbit immunoglobulin (1:2000, Santa Cruz Biotechnology) at 37°C for 2h. Finally, antibody binding wasvisualizedusing electro-chemiluminescence (Thermo Fisher Scientific), and quantified using ImageJ (National Institute of Health, Bethesda, MD, USA).

After electrophoretic separation and immunoblotting of whole-cell lysates, blots were incubated with appropriate primary antibodies and secondary antibodies labeled with horseradish peroxidase. Visualization of proteins was performed using the ChemiDoc XRS system with Image Lab software (Bio-Rad). Antibodies to detect HK2, phosphorylated Akt (Ser473), PARP, caspase-9, c-Myc, phosphorylated GSK3beta (Ser9) were obtained from Cell Signaling Technologies (Danvers, MA, USA). The antibodies against β-actin and LMP1 were from Sigma-Aldrich (St Louis, MO, USA) and DAKO (Glostrup, Denmark), respectively. Anti-α-tubulin, anti-mouse immunoglobulin, anti-rabbit immunoglobulin and normal rabbit immunoglobulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-VDAC, phosphorylated Myc (Thr58) and FBW7 were purchased from Abcam (Cambridge, UK).