Horseradish peroxidase hrp conjugated anti rabbit or anti mouse igg

Cells were lysed in lysis buffer containing 10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate (Na-DOC), 0.1% sodium dodecyl sulfate (SDS), and a protease inhibitor cocktail (Complete EDTA-free protease inhibitor; Nacalai Tesque). Nuclei and membrane fractions were removed by centrifugation. Lysates were separated by SDS-poly-acrylamide gel electrophoresis (SDS-PAGE), and proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with the appropriate primary antibody, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology Inc, Dallas, Texas, USA) secondary antibody. Immunoreactive proteins were visualized by using the Pierce ECL Western Blotting Substrate (Thermo Scientific Pierce, Kanagawa, Japan) and an LAS3000 scanning system (Fuji Film, Tokyo, Japan). The following primary antibodies were used in the immunoblot analysis: anti-VCP (rabbit polyclonal [Cat No. AP6920b], Abgent, San Diego, California, USA; 1:200) and anti-β-actin (mouse monoclonal [Cat No. A5441], Sigma-Aldrich Inc., St. Louis, Missouri, USA; 1:2,000).

GC-1 cells were treated with 5-Aza. TM4 cells and GC-1 cells were transfected with pcDNA 3.1/ZCCHC13. Modified cells were washed with ice-cold Dulbecco’s PBS and then lysed for 30 min on ice in lysis buffer containing protease inhibitor cocktail tablets (Roche, Nutley, NJ, USA). The protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Equal amounts of protein (40 µg) were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel under reducing conditions, after which they were electrophoretically transferred onto PVDF membranes. The blots were then probed with primary antibodies: antibody anti-AKT (1:500), anti-p(Ser129)-AKT1 (1:500), anti-ERK1/2 (1:500), anti-p(Thr202/Tyr204))-ERK1/2 (1:500), anti-CDK4 (1:500), anti-GAPDH (1:1,000), anti-ZCCHC13 (1:400), and anti-c-MYC (1:1,000). All primary antibodies were purchased from Sangon Biotech (Shanghai, China), except for anti-ZCCHC13 antibody (Abcam, ab104509) and anti-c-MYC antibody (Abcam, ab32072). Anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated IgG (Santa Cruz Biotechnology) was then used as appropriate for secondary antibodies at a 1:5,000 dilution; the blots were subsequently developed using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA).