Cab340hu22

The following primary antibodies were used in this study: MS601 (ab110411, Abcam, 1:1000 dilution), NDUFB8 (ab110242, Abcam, 1:1000 dilution), SDHA (ab14715, Abcam, 1:2000 dilution), UQCRC2 (ab14745, Abcam, 1:1000 dilution), COXI (ab14705, Abcam, 1:1000 dilution), ATP5A (ab14748, Abcam, 1:1000 dilution), and β-actin (Cloud Clone Corp. CAB340Hu22, 1:10 000 dilution). HRP-conjugated mouse secondary antibody was used (DAKO, P0260, 1:2000 dilution), followed by detection using the ECL (GE Healthcare) and BioRad imaging system (Image Lab).

After in vitro activation, CD25⁺ or CD25⁻ CD4⁺ cells were harvested and taken up in RIPA buffer with HALT protease and phosphatase inhibitor mix (Thermo Fisher Scientific). Lysates were then incubated on ice for 30 min, spun down at 13,000 g for 30 min at 4 °C and supernatants were transferred to a new eppendorf tube. Protein content was measured using a BCA assay (Pierce), according to manufacturer’s directions. 25 ug of total protein was loaded onto a 12% PAGE gel. Proteins were transferred to nitrocellulose membranes and immunoblotted with primary antibodies against murine HPSE (rabbit polyclonal antibody PAA711Mu04, Cloud-Clone Corp.), human HPSE (mouse monoclonal antibody clone HP3/17, ProSpec), human/murine β-actin (mouse monoclonal antibody CAB340Hu22, Cloud-Clone) and murine GAPDH (mouse monoclonal antibody 6C5, EMD Millipore) and subsequently DyLight-680 or DyLight800-conjugated secondary antibodies (Supplementary Table 2). Antibody binding was visualized and quantified using and Odyssey Imaging system and Image Studio software (Li-Cor).