Anti ddb1

For nuclear lysate preparation, cells were harvested, washed in PBS, and resuspended in Buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol and 0.1% Triton) for 8 min on ice. Nuclei were centrifuged at 1300 × g and resuspended again in Buffer A containing benzonase. To prepare whole cell lysates, cells were harvested, washed and re-suspended in RIPA buffer (50 mM Tris (pH 8), 150 mM NaCl, 0.1% SDS, 1 mM EDTA, 0.5% DOC and 1% NP-40) for 30 min on ice. Nuclei were centrifuged at 12 000 RPM, and the supernatant was collected. Protein concentration was determined using the Bradford Assay (Bio-Rad). All capillary electrophoresis runs were normalized to Lamin (anti-Lamin, Cell Signaling 2032) or total protein (Protein Simple). Primary antibodies used include: anti-Treslin (Bethyl A303-472A), anti-MTBP (Santa Cruz, sc-137201), anti-GFP (Abcam, ab290), anti-CUL4B (Sigma, HPA011880), anti-CUL4A (Cell Signaling, 2699), anti-DDB1 (Santa Cruz Biotechnology, SC-137132), anti-CDT2 (Abcam, ab184548), anti-PCNA (Santa Cruz Biotechnology, PC10, sc-56), anti-CDC45L (Protein Tech 15678-1-AP), anti-WDR5 (Cell Signaling, 13105), anti-CDT1 (Abcam, ab202067), anti-p21 (Cell Signaling, 2947) and anti-SET8 (Cell Signaling, 2996). Images were generated with Compass Software (Protein Simple).

Proteins were extracted from mouse tissue and organoids. 25 micrograms of protein were immunoblotted by standard protocols. The primary antibodies included as the following: anti-CUL4B (Sigma), anti-Lgr5 (BBI Life Sciences, Shanghai, China), anti-Histone (GeneTex), anti-α-Tubulin (Proteintech, Wuhan, China), anti-Cytokeratin 20 (GeneTex), anti-GAPDH, anti-pGSK3β(Ser9), anti-p-β-catenin (S33/37/T41), anti-Non-p-β-catenin, anti-Ub (all antibodies from Cell signaling), anti-IRGM1(GeneTex, Cell signaling), anti-PTGES3 (Proteintech), anti-SLC5A1, anti-ABCG2, anti-STX18 (ORIGENE, Rockville, USA), anti-EPCAM (Abways, Shanghai, China), anti-ACTIN, anti-DDB1, anti-β-catenin (Santa Cruz), anti-HA (Rockland) anti-ROC1 and anti-WDR77, anti-WDR1(Abcam). The secondary antibodies included anti-rabbit and anti-mouse horseradish peroxidase (HRP) (Jackson ImmunoResearch, West Grove, PA, USA; 1:10000; 1:5000). The detection reagent Cheminoluminiscent Substrate was provided from the ECL kit (Thermo, USA). The band signals from Western blot results were analyzed by Volume Analysis of Quantity One with volume background subtraction (Bio-Rad, USA).