Dc lowry

Cells were washed with PBS and lysed in Lysis Buffer [10 mM Tris–HCl, pH 7.4, 1% Triton X-100 containing protease inhibitor cocktail (PIC; Roche)]. Soluble proteins were resolved using SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Protein levels were quantified using the method of DC-Lowry (Bio-Rad), and equivalent amounts of protein were loaded. Uniform protein loading was confirmed using Ponceau S staining of membranes and showed no significant differences in protein levels among samples. Membranes were blocked in 5% milk (w/v) in Tris Buffered Saline (pH 7.4) containing 0.05% Tween 20 (TBS-T), and incubated with antibodies [polyclonal anti-glutaminase (1:10,000), monoclonal anti-GAC/KGA/GAM (1:3000), anti-citrate synthase (1:1000), anti-complex IV subunit 2 (1:1000), anti-β-actin (1:1000), anti-aconitase 1 (1:5000), and anti-TPP (1:10,000) primary antisera]. All antibodies were incubated overnight at 4 °C, except anti-TPP which was incubated for 3h at ambient temperature. Membranes were washed, incubated with HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibody, and developed using SuperSignal West Dura chemiluminescence substrate (Pierce). Images were obtained using an Amersham™ Imager 600 (GE Healthcare Biosciences; Pittsburg PA).