Anti usp24

Human and mouse specimens were incubated in 10% formaldehyde for 72 h for fixation, dehydration, and embedded in paraffin. For immunohistochemistry, xylene (Sigma-Aldrich) was used for dewaxing paraffin-embedded sections and serial diluted ethanol was also used for dehydration. Endogenous peroxidases were blocked by incubating in PBS containing 0.3% hydrogen peroxide (Sigma-Aldrich) for 30 min, and then samples were blocked with 1% bovine serum albumin. Proteins of interest were recognized by incubated with anti-USP24 (Cat#13126-1,-AP, Proteintech, 1:200), anti-p300 (Cat#585, Santa Cruz, 1:100), anti-DNMT1 (Cat#271729, Santa Cruz, 1:100), anti-β-TrCP (Cat#102667, Genetex, 1:100), and anti-NF-ĸB (Cat#8414, Santa Cruz, 1:100) at room temperature for 3 h, and immunoreactivity was visualized by using Vectastain ABC kit (Vector). Sections were photographed by Olympus BX-51 microscope. Specificities of the antibodies for indicated proteins were validated by using the samples from mice and human cohorts and uncropped scans of the blots were shown in Supplementary Fig. 5.

Proteins were electrophoresed by SDS/PAGE and blots were incubated overnight with primary antibody. The following antibodies were used: anti-GPR31 (Abcam, ab75579), anti-ATPase Na⁺/K⁺ β2 (Bioss, bs-1152R), anti-Wnt3a (Cell signaling Technology (CST), #2391), anti-Wnt5a (CST, #2530), anti-pGSK-3β (phospho Ser9, CST, #5558), anti-JNK (phospho Thr183/Tyr185, CST, #4671), anti-β-catenin (CST, #8480), anti-HA tag (CST, #5017), anti-flag tag (CST, #14,793), anti-USP24 (Proteintech, 13,126–1-AP), anti-FGGY (Abnova, ABN-H00055277), anti-β-Actin (CST, #4967), and anti-GAPDH (Abcam, ab181602). After incubated with HRP-conjugated α-rabbit or α-mouse secondary antibodies for 1 h, protein bands were detected with chemiluminescence substrate (Perkin Elmer) using the ChemiDoc Imaging System (Bio-Rad).

Cells were collected by sample buffer and analyzed by electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF, Millipore) membrane and TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.05% Tween 20) containing 5% nonfat milk was used for blocking. Anti-USP24 (Proteintech), anti-p300 (BD), anti-actin (Sigma-Aldrich), anti-ubiquitin (Santa Cruz), anti-P-gp (Genetex), anti-ABCG2 (Genetex), anti-MRP1 (Genetex), anti-MRP3 (Genetex), anti-Ezrin (Genetex), lamin A/C (Santa Cruz), anti-γ-H2Ax (abcam), anti-biotin (Genetex), anti-Flag (Genetex), anti-Rad51 (abcam), and anti-BRD7 (Sigma-Aldrich) were used for probing interested proteins. After incubated with primary antibodies, PVDF membranes were then incubated with secondary immunoglobulin antibodies linked with horse radish peroxidase (Millipore, 1:10,000). For detecting immunoprecipitated samples, light chain-specific secondary antibodies were used (Jackson ImmunoResearch, 1:10,000). ECL Western blotting detection system (Millipore) and ChemiDoc-it imager (UVP) were used for detecting signals.