Beyoecl star

The samples were mixed with 5× SDS-PAGE sample loading buffer and boiled for 5 min. Protein bands were separated by SDS-PAGE with a 12% separating gel and a 5% stacking gel, and transferred to nitrocellulose membranes (Beyotime, Shanghai, China). The membranes were incubated with 5% nonfat dried milk in TBS with 0.1% Tween-20 (TBST) for blocking, and then incubated with 6 × His-tag mouse monoclonal antibody (1:2500 dilution, EnoGene Biotech, Nanjing, China). After washing with TBST, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000 dilution, EnoGene Biotech, Nanjing, China). Finally, the immunoreactive proteins were detected with BeyoECL Star (Beyotime, Shanghai, China).

Tissue collected from the anus and rectum of the ARM model and normal rat embryos was pooled and sonicated in double-distilled H₂O containing protease inhibitors. Protein extracts (50 μg) were denatured by heating at 90°C for 5 min and then stored at −80°C refrigerator until used. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime, Shanghai, China), transferred to the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), blocked with 5% fat-free milk in Tris-buffered saline for 1.5 h at room temperature, and incubated overnight at 4°C with primary anti-Fgf9 rabbit polyclonal antibody (Abcam, Cambridge, UK; 1 : 1000 dil). The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Beyotime, Shanghai, China; 1 : 1000 dil) for 2 h at room temperature. A chemiluminescent substrate kit (BeyoECL Star; Beyotime, Shanghai, China) was used to detect the immunostained bands. In each lane, β-actin was used as an internal standard to normalize protein expression.

Briefly, isolation of total protein from liver tissues was performed using RIPA Lysis Buffer (Beyotime, Shanghai, China) added with Protease-Phosphatase Inhibitor Cocktail (Beyotime, Shanghai, China) on ice, and the supernatants were collected after centrifugation. Protein concentrations were measured using a BCA Protein Assay kit (Beyotime, Shanghai, China). Equal proteins of each group were loaded on 10% SDS-PAGE to separate them and were delivered into polyvinylidene difluoride membrane. The membranes were blocked in 5% skimmed milk, which dissolved in Tris buffered saline with Tween 20. Then, they were incubated with primary antibodies against Cleaved-GSDMD (10,137; 1:1000; CST), β-actin (AF5003; 1:5000; Beyotime) overnight at 4 °C. After being incubated with secondary antibodies (A0208; 1:1000; Beyotime), the blots were visualized with enhanced chemiluminescence BeyoECL Star (Beyotime, Shanghai, China), and the chemiluminescent signals were captured using the ChemiDoC XRS+ system. Finally, the relative intensity of the chemiluminescent signals was quantified using the ImageJ software.

The fluorescence spectra were acquired on a SPEX Fluorolog 3-TCSPC spectrometer equipped with 1 cm path length cuvettes. The pH value was measured by PHS-3C Precision Ph/Mv Meter. The cell images were acquired on an Olympus Biological Microscope System. The 3 peptides, TAM-PEP (R4): TAM-RRRRNLWAAQRYGRELRRMSDKFVD, TAM-PEP (R6): TAM-RRRRRRNLWAAQRYG RELRRMSDKFVD and TAM-PEP (R8): TAM-RRRRRRRRNLWAAQRYGRELRRMSDKFVD, were synthesized by GL Biochem (Shanghai, China) Ltd. GO (XF-020, 50–200 nm) was purchased from XianFeng Nano Co. (Nanjing, China). The cDNA3.1-Bcl-xL plasmid was constructed by Genecreate Biological Engineering Co. (Wuhan, China) Tris-HCl, IPTG, PMSF, Anti-Bcl-xL antibody, Anti-ACTB antibody, HRP-conjugated Goat Anti-Rabbit IgG, Cell Counting Kit-8, Cytochrome C, BSA, RNase and lysozyme were purchased from Sangon Biotechnology Co. (Shanghai, China) BeyoECL Star and trypsin digestion solution were purchased from Beyotime Biotechnology Co. (Shanghai, China) Lipo ™ 2000 was purchased from Thermo Fisher Scientific Co.(Beijing, China)The deionized water was prepared on a Milli-Q water purification system and used throughout all experiments.

Western blot was performed according to the previous study (29 (link)). The proteins were extracted from the striatum and the concentrations were determined by the BCA Protein Assay kit (Thermo Scientific, United States). The protein (30 μg) was loaded onto 12% SDS-PAGE (Solarbio, Beijing, China) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, United States). The blot was incubated in blocking buffer (5% milk in Tris-buffered saline with 0.1% Tween-20) and then incubated with rabbit anti-MBP (Cat# ab40390, Abcam, United States; 1:1,000) or rabbit anti–β-actin (1:1,000, Catalog No. 4970S; Cell Signaling Tech) overnight at 4 °C. After washing with TBST (0.01% Tween 20 in TBS), the blots were incubated with horseradish peroxidase-conjugated IgG secondary antibody (Biosharp, BL003A, 1:10,000) for 1 h at room temperature and then reacted with BeyoECL Star (Beyotime, Shanghai, China). The chemiluminescence results were recorded by Amersham Imager 680 imaging system (CTL, United States) and analyzed by ImageJ (NIH, United States).

Conventional western blotting was performed following a standard protocol [20 (link)]. The following antibodies and dilutions were applied: anti-UCHL1 (1: 1000, 14730-1-AP/66230-1-Ig, Proteintech), anti-KLF5 (1:1000, 66850-1-Ig/21017-1-AP, Proteintech), anti-Ubiquitin (anti-Ub, 1:1000, 10201-2-AP, Proteintech), anti-Flag tag (1:1000, 66008-4-Ig, Proteintech), anti-Myc tag (1:1000, 60003-2-Ig, Proteintech), anti-TET1 (1:1000, 61,443, Proteintech, Wuhan, China), anti-TET3 (1: 1000, ABE290, Merck, Darmstadt, Germany) and anti-β-actin (1:2000, 20536-1-AP, Proteintech). Then, the protein bands were visualized using Enhanced chemiluminescence (ECL) (BeyoECL Star, Beyotime, Shanghai, China).

Following TB treatment, HOG and U251 cells were harvested and washed twice with cold PBS. Then, the cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (P0013B, Beyotime, CN) containing protease inhibitor (C600386, Sangon Biotech, CN) and phosphatase inhibitor (C500017, Sangon Biotech, CN) for 30 min on ice, with repeated freezing and thawing for three times. The lysates were then sonicated and centrifuged at 12,000 rpm for 15 min at 4°C to obtain the total protein supernatant. Protein concentrations were determined by a commercial BCA kit (P0010, Beyotime, CN). Appropriately 20 μg of lysed protein was boiled in sample-loading buffer and separated by a denaturing 10% SDS-PAGE. The gels were transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States) and blocked with 5% nonfat milk for 1.5 h, followed by overnight incubation at 4°C with the following primary antibodies: c-Myc, cyclin A1, cyclin B1, cyclin D1, P53, p-P53, Cdk1, Cdk2, Cdk4, AKT, p-AKT, Erk, p-Erk, active caspase-3, Bax, Bcl-2, PTEN, and GAPDH. Following the incubation with peroxidase-conjugated goat anti-rabbit IgG or anti-mouse IgG at room temperature for 1 h, the membrane was visualized using BeyoECL Star (BEYOTIME, China), and pictures were taken using a ChemiDoc imaging system (BIO-RAD, United States).