Ab109029

Lung cancer tissues and paracancerous tissues were embedded by paraffin and sectioned into 4‐μm‐thick sections. Paraffin‐embedded tissue sections were deparaffinized and hydrated. Citric acid (pH 6) was used to extract antigens for 30 minutes at 97°C after which the antigens were treated with 3% H₂O₂. Then, the tissue sections were incubated with the anti‐rabbit antibodies against FXYD3 (ab205534, 1:400, Abcam), KDM3A (ab191387, 1:500, Abcam) and DCLK1 (ab109029, 1:500, Abcam) overnight at 4℃. Afterwards, the sections were incubated with the horseradish peroxidase (HRP)‐conjugated secondary antibody (Dako; Agilent Technologies, Inc) for 30 minutes. Following 10‐minute incubation with 3,3'‐diaminobenzidine tetrahydrochloride, the sections were re‐stained with haematoxylin for 2 minutes. Finally, the pathology results were obtained under a microscope.

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.