Flag 80010 1 rr

Human MT4 T cells and Jurkat cells were grown in Roswell Park Memorial Institute (RPMI) 1640 (Gibco, Logan, UT, USA) at 37°C in 5% CO₂. HOS.CD4.CCR5 cells (hereinafter referred to as HOS cells) and 293T cells were grown in complete Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Logan, UT, USA) supplemented with 10% heat-inactivated fetal calf serum at 37°C in 5% CO₂. The cells were subsequently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions or inoculated with HIV-1 as described previously [25 (link)]. All cells were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China).
Antibodies against HIV-1 p24 (sc-69728) and phosphor-p50 (p-p50; sc-271908) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p50 (14220-1-AP), Actin (66009-1-Ig), LaminB (12987-1-AP), and Flag (80010-1-RR) were purchased from Proteintech (Rosemont, IL, USA). Antibody against TGIF2 (ab155948) was purchased from Abcam (Cambridge, MA, USA). Antibody against Vpr (MA-001-2) was purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan).

The radio immunoprecipitation assay lysis buffer (RIPA) (P0013B, Beyotime, China) containing a protease-inhibitor cocktail (HY-K0010, MCE, USA) were used to extract proteins from cells. The proteins were lysed in SDS-loading buffer (FD006, Fdbio, China), then 20μg proteins were resolved on a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA). The membrane was incubated with primary antibodies against PD-L1(13684, Cell Signaling Technology, USA), FLAG (80010-1-RR, Proteintech, USA), STT3A (12034-1-AP, Proteintech, USA), c-Jun (AF6089, Affinity, USA), HA (51064-2-AP, Proteintech, USA) and GAPDH (60004-1-Ig, Proteintech, USA) at a dilution of 1:1000, followed by incubation with species-specific (rabbit or mouse) HRP-conjugated secondary antibodies at a dilution of 1:5000. The immunoreactive bands were visualized by Omni ECL reagent (SQ101, EpiZyme, China).