Anti pdh antibody

PDH protein was quantified in lysed pellets of FACS-purified iNs using the ProteinSimple Jess (Biotechne) and the 12-230 kDa Jess Separation Module in the NIR channel. Pellets were lysed in RIPA Lysis and extraction Buffer (ThermoFisher) containing cOmplete EDTA-free Protease Inhibitor Cocktail (Merck, 11873580001) and PhosSTOP (Merck, 4906845001). Anti-PDH antibody (Santa Cruz, sc-377092, 1:50) was incubated for 60 minutes, followed by standard default run settings provided by ProteinSimple. Data analysis was performed using Compass software.

Whole-cell protein was isolated using RIPA buffer according to manufacturer’s protocol (Sigma-Aldrich). Protein was then blotted using 12% Bio-Rad precast gels and PVDF membranes. To indicate protein lipoylation levels, an anti-lipoate antibody was used. To detect levels of electron transport chain proteins, an OXPHOS antibody cocktail (MitoSciences MS604) was used. To indicate the presence of ER contamination in isolated mitochondria samples, an antibody against FACLS4 was used (Santa Cruz), and an anti-PDH antibody (Santa Cruz) was used for protein normalization. These blots indicated that mitochondrial samples were free from ER contamination (data not shown).