Anti gapdh antibody

Protein was extracted from cells lines using RIPA lysis buffer with proteinase inhibitors (Thermo). The Protein BCA Assay Kit (Thermo) was used to measure the concentration of protein in the lysates. Each lane was loaded with 20 µg protein mixed with 2× SDS loading buffer (Thermo). The protein was separated by 12% SDS‐polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore). The membranes were incubated with 5% skim milk powder at room temperature for 1 hour to block non‐specific binding. Subsequently, the membranes were incubated with antiserum containing antibodies against Jagged1, ZEB1 and Bmi1 (CST), at 4°C for 12 hour. To visualize the target proteins, a 1:5000 dilution of peroxidase‐conjugated secondary antibody and ECL Western blotting detection reagents (GE) were used. A Bio Image Intelligent Quantifier 1‐D was used to quantify the proteins (Version 2.2.1, Nihon‐Bio Image Ltd). An anti‐GAPDH antibody was used as a protein loading control (Boster).

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.