Anti mct1

CD4⁺ T cell protein extracts (~ 30 μg) were boiled in Laemli sample buffer (Sigma-Aldrich) for 5 min, resolved on 10% SDS-polyacrylamide gels and transferred onto PVDF filters (Amersham). Membranes were blocked for 1 h in Tris-buffered saline (TBS), 0.05% Tween-20, 5% non-fat dry milk, followed by overnight incubation with specific primary antibodies diluted in the same buffer. The primary antibodies used are listed as follows: anti-GLUT1 (1:1000, Novus Biologicals), anti-MCT1 (1:1000, Novus Biologicals), anti-β-actin (1:8000, Sigma-Aldrich). Mitochondria-rich pellet (15 μg) was separated on a SDS-PAGE and, after blocking, membranes were incubated with MitoProfile total OXPHOS human WB antibody cocktail (1:1000, Abcam) and anti-Porin (1:1000, Santa Cruz). After washing with 0.1% Tween in TBS, membranes were incubated with a peroxidase-conjugated secondary antibody for 1 h, washed and developed using the ECL chemiluminescent detection system (Clarity™ Western ECL Substrate Biorad). The densitometric analyses of blots were performed by a computerized image processing system (Image J, 1.0 version).