Immunoblotting Analysis of Cellular Proteins

Total crypt cellular or nuclear extracts (30–50 μg protein/lane), were subjected to SDS-PAGE and electrotransferred to nitrocellulose membrane. The membranes were blocked with 5% BSA or 5% nonfat dried milk in Tris-buffered saline (TBS) (20 mM Tris-HCl and 137 mM NaCl, pH 7.5) for 1 h at room temperature (21 °C). Immunoantigenicity was detected by incubating the membranes overnight with the appropriate primary antibodies (0.5-1.0 μg/ml in 5% BSA or 5% nonfat dried milk) and Western blot analysis was performed as described [10 (link)]. Antibodies used were anti-DCLK1 (1:1000, ab31704, ab109029) from Abcam, anti-p62/SQSTM1 (1:400, H00008878-M01), anti-LC3B (1:300, NB100-2220) from Novus Biologicals, anti-GAPDH(1:3000), anti-β-actin (1:5000), from Sigma-Aldrich, anti-EZH2 (1:000), anti-H3 (1:1000) were from Cell Signaling Technology.