Protein a g coupled agarose

For immunoprecipitation, cells were lysed in HEPES-lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl₂, 5 mM EDTA, 10% glycerol and 1% Triton X-100, pH7.4) supplemented with a protease inhibitor cocktail (Selleck Chemicals), and then centrifuged at 14,000 g for 15 min. For IP reactions with ectopically expressed and tagged proteins, Flag- (Selleck Chemicals) or HA-beads (MCE) were added to the lysates, stirred at 4 °C for 2 h and washed with HEPES-lysis buffer 4 times. Immuno-precipitated proteins were eluted with Flag or HA peptide (MCE) dissolved in 100 ul HEPES-lysis buffer for 3 h, and the supernatant collected for analyses. For IP reactions with endogenous G6PD, the supernatants were pre-cleared with protein A/G-coupled agarose (Thermo Fisher Scientific), anti-G6PD antibody was added to the cell lysates and stirred at 4 °C overnight, and then protein A/G-coupled agarose were added and incubated for 2 h, followed by washing 4 times and boiling in 5X loading buffer. Immuno-enriched samples were resolved by SDS-PAGE and analyzed by Western-Blot.