Bmal1

For immunoblot analysis, isolated colon crypts or YAMC cells scraped off from the dish were lysed with NP40 lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% NP40, pH 8.0) supplied with protease inhibitor cocktail (Roche, USA) on ice for 20 min before 5 min 6000 rpm centrifugation at 4°C. Supernatants were collected for SDS-PAGE analysis. The primary antibodies were incubated overnight at 4°C.
Antibodies: GR/NR3C1 (Cell Signaling Technology, Cat.3660, 1:1000), REV-ERBα /NR1D1(Abcam, Cat. ab174309, 1:1000), NLRP3(Cell Signaling Technology, Cat.15101, 1:1000), IL1β(Abclonal, Cat.A16288, 1:1000), IL6 (Cell Signaling Technology, Cat.12912, 1:1000), CLDN1 (Thermo Fisher, Cat.37-4900, 1:10000), ORMDL3 (Abclonal, Cat.A14951, 1:2000), NPAS2 (Abclonal, Cat.A16930, 1:2000), PARD3 (Proteintech, Cat.11085-1-AP, 1:500), ACTB/β-actin (Abclonal, Cat.AC026, 1:10000), BMAL1 (Proteintech, Cat.14268-1-AP, 1:5000), CLOCK (Abclonal, Cat.A7265, 1:10000), Caspase-1 (Proteintech, Cat.22915-1-AP, 1:1000), IFIT3 (Abclonal, Cat.A3924, 1:1500), CDH1 (Abclonal, Cat.A11492, 1:1500), CDH3 (Abclonal, Cat.A14235, 1:1500).

Total proteins from cells were extracted with RIPA lysis buffer containing 1 mM PMSF (Fude: http://www.fdbio.net) and phosphatase inhibitor (Fude). Protein concentration was measured using a BCA Protein Assay kit (CWBio: http://www.cwbiotech.bioon.com.cn). The proteins (50 µg/lane) were loaded on 10% SDS-PAGE and separated by electrophoresis, followed by blotting on a PVDF membrane (EMD Millipore). The membrane was blocked in Tris-buffered saline (pH 8.0)+0.1% Tween-20 and 5% skim milk at room temperature for 2 h. The corresponding primary antibody (1:1,000) was incubated overnight at 4°C and then incubated with the horseradish peroxide-conjugated secondary antibody (Fude; 1:10,000) at room temperature for 1 h. Immunological signals were detected using an electrochemical luminescence kit (Yeasen; www.yeasen.com). The band intensities were quantified using Image-Pro-Plus 6.0 software (MediaCybernetics, Inc.). The primary antibodies are as follows: CRY1 (cat. no. ab245564; Abcam), PER2 (cat. no. ab179813; Abcam), CLOCK (cat. no. 18094-1-AP; ProteinTech Group, Inc.), BMAL1 (cat. no. 14268-1-AP; ProteinTech Group, Inc.), p53 (cat. no. 2524; Cell Signaling Technology, Inc.), p21 (cat. no. 10355-1-AP; ProteinTech Group, Inc.) and GAPDH (cat. no. AP0063; Biogot Technology Co., Ltd.).