Ptm 105rm

The cells and mouse tissues were lysed using RIPA buffer (#89901, Thermo fisher Scientific) supplemented with PhosSTOP and complete protease inhibitors (Merck, Beijing, China). The protein samples were subjected to 12.5% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (162-0177, BioRad, Shanghai, China). Blots were probed with appropriate primary antibodies against VCAM-1 (ab134047, Abcam, Cambridge, UK), E-selectin (20894-1-AP, Proteintech, Shanghai, China; sc-137054, Santa Cruz, California, USA), Phosphorylated ACLY (Ser455, 4331, CST, Danvers, MA), ACLY (13390, CST), Raptor (48648, CST), Rictor (9476, CST), phosphorylated FoxO1 (Ser256, 84192, CST), FoxO1 (2880, CST), acetylated FoxO1 (Lys294, AF2305, Affinity, PA, USA), MYC (9402, CST), FASN (3180, CST), ACSS2 (3658, CST), ACC1 (4190, CST) (MA, USA), and pan-acetyl-lysine (PTM-105RM, PTM Biolabs, Shanghai, China) at 4 °C overnight. The blots were then incubated with the appropriate secondary antibodies (1:5000, proteintech) and detected using a horseradish peroxidase substrate (WBLUF0500, Millipore, USA). For internal controls of equal loading, the blots were also stripped with stripping buffer (100 mmol/l 2-mercaptoethanol, 2% SDS, 62.5 mmol/l, Tris pH 6.8) and reprobed with ACLY, FoxO1, or GAPDH antibodies.

The cell lysates were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Millipore). Subsequently, the membranes were blocked with 5% skimmed milk and incubated overnight at 4 °C with primary antibodies. The following antibodies were used: anti-SIRT5 (1:1000 dilution; 8782 S, Cell Signaling Technology, Danvers, MA, USA), anti-HINT1 (1:1000 dilution; 67583-1-Ig, Proteintech, Rosemont, IL, USA), anti-Succinyllysine (1:1000 dilution; PTM-401, PTM Bio, Hangzhou, China), anti-Acetyllysine (1:500 dilution; PTM-105RM, PTM Bio), anti-GFP (1:5000 dilution; 50430-2-AP, Proteintech), anti-Flag (1:5000 dilution; 80010-1-RR, Proteintech), and anti-β-Actin (1:5000 dilution; AB0035, Abways, Shanghai, China). Then PVDF membranes were incubated with HRP-conjugated goat anti-rabbit or mouse antibodies (1:5000 dilution; AB0101 and AB0102, Abways) and thoroughly washed. The protein bands were visualized using the ECL chemiluminescence detection kit (WBKLS0500, Millipore), and band intensity was quantified using ImageJ software.