Fusion fx edge imaging system

Cells were lysed with T-PER protein extraction reagent (Thermo Fisher Scientific) containing a protease and phosphatase inhibitors cocktail. Proteins were separated by SDS-PAGE and transfered to PVDF membranes. After blocking with 5% bovine serum albumin for 1 h, the membranes were incubated with primary antibodies (1:1000 dilutions) for Arg1, CD206, SIRT5, PDHA1, Pan-succK, Pan-acetK and tubulin at 4°C overnight, followed by incubation with HRP-linked secondary antibodies (1:2000 dilutions) at room temperature for 1 h. Chemiluminescence images were visualized using a Super ECL plus western blotting Substrate kit (Bioground biotechnology, Chongqing, China) and detected using a Vilber Fusion FX-EDGE imaging system (Grand Paris, France).

Dynabeads protein A/G (5 μg; Thermo Fisher Scientific) were mixed with 10 μg of anti-SIRT5 or anti-PDHA1 antibody (Santa Cruz Biotechnology, Heidelberg, Germany) and incubated at room temperature with rotation for 30 min. Then 250 μg of cytoplasmic proteins were added and incubated overnight at 4°C. The dynabeads were washed three times with washing buffer. The proteins bound to the dynabeads were eluted with acid elution buffer and then boiled at 100°C for 5 min in loading buffer and separated by SDS-PAGE. The proteins were transferred to a PVDF membrane and blocked with 5% bovine serum albumin for 1 h. The membrane was incubated with anti-PDHA1, anti-SIRT5 and anti-pan-succinyllysine primary antibodies (1:1000 dilutions) at 4°C overnight. The membrane was washed with TBST and incubated with an HRP-linked secondary antibody (1:1000 dilutions) for 1 h. After washing with TBST, the bands were visualized with a Super ECL plus western blotting Substrate kit (Bioground, Chongqing, China) and detected using a Vilber Fusion FX-EDGE imaging system (Paris, France).