Anti mouse igg conjugated to hrp

TatC (lack Ser46Phe) and Tat variants (TatN12, TatD60, TatVT6) were cloned into: (a) mammalian expression vector pCMV-myc vector (Clonetech) under the CMV promoter for functional studies, and (b) prokaryotic expression vector pGEX-4T-2 (Invitrogen) to obtain GST-tagged proteins. HIV-1 subtype B TAR was cloned in pcDNA3.1 (Invitrogen) for TAR synthesis to determine Tat–TAR binding activity. Anti-Tat antibody (NIH AIDS Reagent Programme), Anti-myc antibody (Clontech), Anti-GAPDH antibody (Cell Signaling Technology), Anti-rabbit IgG conjugated to HRP (Jackson Immunoresearch), and Anti-Mouse IgG conjugated to HRP (Jackson Immunoresearch) were used in western blotting.

HEK-293T (Human Embryonic Kidney 293 cells; NIH AIDS Reagent Programme) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 100 units penicillin, 0.1 mg streptomycin and 0.25 μg amphotericin B per ml at 37 °C in the presence of 5% CO₂. All transfections were performed using Lipofectamine 2000 (Invitrogen) reagent. pCMV-Myc-Vif, pCMV-HA-Ub, pCMV-Myc-A3G were used in the experiments. Anti-Myc monoclonal antibody (Clontech), anti- HA tag polyclonal antibody (Clontech), anti-GAPDH antibody (Cell Signalling Technology), anti-vif monoclonal antibody (NIH AIDS Reagent Programme), anti-Rabbit IgG conjugated to HRP (Jackson Immunoresearch), anti-Mouse IgG conjugated to HRP (Jackson Immunoresearch) were used in the experiments.

Anti-c-myc monoclonal antibody (Clontech, USA; 1:1,000), anti-GAPDH antibody (Cell Signaling Technology, USA; 1:10,000), anti-6X-His monoclonal antibody (Sigma; 1:2,000), anti-p24 monoclonal antibody (NIH AIDS Reagent Program, USA; 1:3,000), anti-PARP(Cell Signaling Technology, USA; 1:1000), anti-Rabbit IgG conjugated to HRP (Jackson Immunoresearch, USA; 1:10,000) and anti-Mouse IgG conjugated to HRP (Jackson Immunoresearch; 1:10,000) were used for the western blotting experiments.