Anti gapdh

Cells were washed with PBS, collected, and lysed on ice for 30 min with modified Radioimmune Precipitation Assay (RIPA) Buffer (Applygen) containing a protease inhibitor cocktail (Amresco). Cell lysates were then centrifuged for 15 min at 12,000 × rpm and 4°C. The supernatant was collected, and the protein concentration was measured using the BCA Protein Assay Reagent (Pierce). Total protein (30-80 μg) was subjected to 10–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and was transferred to nitrocellulose membranes (Millipore). After blocking in 5% non-fat dry milk in TBST (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% Tween20), the membranes were incubated with primary antibodies overnight at 4°C. After washing in TBST buffer, membranes were incubated for 1 h in the dark with the appropriate IRDye TM 800-conjugated secondary antibodies (Thermo) in TBST/5% non-fat milk. Signals were detected on an Odyssey Infrared Imager (LI-COR Bioscience) after washing.
The following antibodies were used for the Western blot analysis: anti-SIRT6 (Abcam, CST), anti-Multi Ubiquitin (MBL), anti-FLAG (Sigma), anti-Myc (MBL), anti-p27 (MBL, Santa), anti-GAPDH (Tianjin Sungene Biotech Co., Ltd.), anti-β-TUBULIN (Santa), anti-p21 (MBL), anti-p53 (Santa), anti-p16 (Santa), anti-PTEN (Santa), anti-acetylated lysine (CST).

The protein was separated by 1×sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We transferred the protein to 0.22 μm PVDF membrane by using wet transfer. After being blocked in 8% non-fat milk overnight, the membrane were incubated by primary antibodies including anti-Pcna (1:1000, Mouse IgG, Cell Signaling Technology), anti-C-Myc (1:1000, Rabbit IgG, Cell Signaling Technology), anti-P53 (1:500, Rabbit IgG, Cell Signaling Technology), anti-caspase3 (1:500, Rabbit IgG, Beijing Biosynthesis Biotechnology) and anti-β-tublin (1:1000, Mouse IgG, Sino Biological), anti-Sirt1 (1:500, Rabbit IgG, Sangon Biotech), anti-A-β-catenin (1:1000, Rabbit IgG, Abcam), anti-PDX1 (1:1000, Rabbit IgG, Sangon Biotech), anti-β-catenin (1:500, Mouse IgG, Santa Cruz), anti-Acetyl-β-catenin (lys49) (1:1000, Rabbit IgG, Cell Signaling Technology), anti-GAPDH (1:1000, Mouse IgG, Tianjin Sungene Biotech) at 4°C for 12 h. Horse-radish peroxidase-conjugated anti-rabbit and anti-mouse (1:1000, Beyotime) treated the membrane at 37°C for 1 h. The blots were viewed and analyzed by Tanon-4200 (Shanghai Tianneng Corporation, China).

Macrophages (5 × 10⁵ cells/mL) were stimulated with various doses of ES (0.1 μg/mL, 1 μg/mL, and 10 μg/mL), LPS (100 ng/mL), and Pam3CSK4 (4 μg/mL) for different time points. The treated cells were washed twice with PBS and then lysed for 30 min on ice in a RIPA solution containing a protease inhibitor cocktail and phosphatase inhibitors (Sigma, USA). The expression of proteins in the cell lysates were examined using anti-NF-κB phospho-p65, anti-phospho-JNK, anti-phospho-p38, and anti-phospho-ERK1/2 antibodies (Cell signaling technology, USA). Anti-GAPDH (Sungene Biotech, China) was used as an internal control. Statistical analysis was performed for band intensities and evaluated using image J (NIH, USA).