Ab86002

Total protein was extracted from tissue or cell by radioimmunoprecipitation assay (RIPA) lysate containing PMSF (P0013C, Beyotime, Shanghai, China) followed by protein concentration detection. The samples were then transferred to a polyvinylidene fluoride membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After that, the membrane was blocked with 5% skim milk powder at room temperature for 1 h and incubated overnight at 4 °C with diluted primary rabbit antibodies against ELK4 (1:1000, ab86002, Abcam, Cambridge, MA, USA), KDM5A (1:5000, ab194286, Abcam), PJA2 (1:800, ab131118, Abcam), KSR1 (1:1200, ab68483, Abcam), and GAPDH (ab9485, Abcam; internal reference). The membrane was then incubated with HRP-labeled goat anti-rabbit against IgG H&L (ab97051, 1:2000, Abcam) for 1 h. Subsequently, the membrane was developed by the enhanced chemiluminescence kit (BB-3501, Ameshame, UK). The images were taken by Bio-Rad image analysis system (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed by Quantity One v4.6.2 software.

Protein extraction from cells and tumor tissues and western blot analysis were performed as described as previously described [12 (link)]. Antibodies against ELK4 (abcam, ab86002), EZH2 (abcam, ab191250), H3K9me1 (abcam, ab16989), H3K9me2 (abcam, ab1220), H3K9me3 (abcam, ab8898), H3K27me2 (abcam, ab24684), H3K27me3 (abcam, ab6002), H3 (abcam, ab1791), E-cadherin (CST, 14472), EAF2 (CST, 14159), ADRB2 (abcam, ab182136), rap1GAP (abcam, ab32373), RUNX3 (abcam, ab224641), GAPDH (CST, 5174) and Notch1 (CST, 3608) were used.