Peroxidase conjugated secondary antibody

Protein was extracted from gastric cancer cell lines using RIPA lysis buffer containing proteinase inhibitor. The protein concentration of the lysates was measured with the Protein BCA Assay Kit (Bio-Rad, USA). For the Western blot assay, 20 µg of protein mixed with 2×SDS loading buffer was loaded per lane, separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). To block nonspecific binding, membranes were incubated at room temperature for 1 h with 5% skim milk powder, followed by a 12-h incubation at 4°C with antiserum containing antibodies against c-Src (B-12) (Santa Cruz Biotechnology sc-8056, Santa Cruz, CA, USA), p44/42 MAPK (Erk1/2; Thr202/Tyr204), total ERK, AKT, pRaf, and β-actin (Cell Signaling Technology, Beverly, MA, USA). Ras activity was detected using the Ras Activation Assay Kit from Upstate-Millipore (17–218). A peroxidase-conjugated secondary antibody (1∶5000 dilution) and ECL Western blotting detection reagents (New England Biolabs, USA) were used to visualize the target proteins, which were quantified with a Bio Image Intelligent Quantifier 1-D (Version 2.2.1, Nihon-BioImage Ltd., Japan). An anti-β-actin antibody (Boster, Wuhan, China) was used as a protein loading control.

Protein was extracted from CRC cell lines using RIPA lysis buffer containing a proteinase inhibitor. The protein concentration in the lysates was measured with the Protein BCA Assay Kit (Bio-Rad, USA), and 20 μg of protein mixed with 2 × SDS loading buffer was loaded in each lane. The proteins in the lysates were then separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). To block nonspecific binding, membranes were incubated at room temperature for 1 h in 5% skim milk. Next, the membranes were incubated for 12 h at 4°C with an antiserum containing antibodies against DNMT1, DNMT3A, DNMT3B, SP1, E-cadherin, MGMT or P16, which were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). A peroxidase-conjugated secondary antibody (1:5000 dilution) and ECL western blotting detection reagents were used to visualize the target proteins (ECL New England Biolabs, USA), and the resultant signals were quantified with a Bio Image Intelligent Quantifier 1-D (Version 2.2.1, Nihon-BioImage Ltd., Japan). An anti-GAPDH antibody (Boster, China) was used as a protein loading control.