Anti ctla 4

Cells were collected and lysed with NP40 Lysis Buffer (Beyotime) with 1 mM Phenylmethanesulfonyl fluoride (Beyotime). Each quantity of 10⁶ cells was lysed with 50 μL NP40 buffer. The cell lysates were collected by centrifugation at 13,000 rpm at 4°C for 10 minutes. Protein loading buffer (Beyotime) was added, and the samples boiled at 95°C for 10 minutes. The isolated protein was subjected to SDS-PAGE on 4% to 20% polyacrylamide gels (ACE Biotechnology, Nanjing, China) and transferred to NC membranes (Millipore, St. Louis, MO, USA). The membranes were incubated with a primary antibody and secondary antibody. The primary antibodies included anti-Glut1, anti-LDHA, anti-NRF1, anti-NRF2, anti-GZMB, anti-Actin (Beyotime), anti-SIRT1 (Abclonal, Wuhan, China), anti-HK2, anti-CPT1α, anti-PGC1, anti-MTCO2, anti-TFAM, anti-GZMA, anti-GZMK, anti-PRF1, anti-PD-1, anti-TIM-3, anti-CTLA-4, anti-NDUFB8, anti-SDHB, anti-MTCO1, anti-UQCRC2, and anti-ATP5A (Abcam, Cambridge, UK). The secondary antibodies included HRP-labeled goat anti-rabbit and HRP-labeled goat anti-mouse antibodies (Beyotime). Finally, the proteins were detected by West Femto Maximum Sensitivity Substrate (ThermoFisher).

The following antibodies were used: anti-CTLA-4 ipilimumab mAb (Yervoy, Bristol Myers Squibb, NY, USA), anti-PD-L1 Atezolizumab mAb (InvivoGen, San Diego, CA, USA); commercial human anti-PD-L1 (G&P Biosciences, Santa Clara, CA, USA) and anti-CTLA-4 (R&D Systems, Minneapolis, MN, USA) Abs; anti-human IgG (H+L) HRP-conjugated Ab (Promega, Madison, WI, USA); anti-human IgG (Fab’)2 goat HRP-conjugated monoclonal antibody (Abcam, Biomedical Campus, Cambridge, UK).
ID-1 (anti-CTLA-4) and PD-L1_1 (anti-PD-L1) mAbs were produced and purified, as previously described [32 (link),33 (link),34 (link),35 (link)], taking advantage of the enhanced cell line HEK293_ES1 expressing a long non-coding SINEUP RNA [36 (link),37 (link)].