Anti samhd1

THP-1 and U937 cells were transfected with pSIVmac, pHIV-2-Patient, pHIV-2-NIH and mock plasmid, and then incubated for 24 h. Proteins were extracted by using cell lysis buffer (Radio immunoprecipitation assay (RIPA)) (Sigma-Aldrich cat no. R0278) containing protease cocktail inhibitors (Sigma-Aldrich cat no. P8340). After measuring the concentration at 595 wave lengths, 50 μg of proteins and pre-stained protein ladder (Bio rad cat no. 1610377) were separated by electrophoresis and transferred to the PVDF membrane. Anti-SAMHD1 (1:500) (Abcam cat no. ab83982), anti-Vpx (1:500) (Mybiosource cat no. P06939) and anti-β-actin (1:1000) (Cell Signaling; cat no. 8H10D10) antibodies were used to probe the western blot.

Proteins were extracted using RIPA buffer (Sigma), supplemented with protease (1:1000, Sigma) inhibitor cocktails, after 30 min incubation on ice. Protein concentrations were measured using BCA assay, with BSA for the standard curve, and 50 μg of protein were resolved on 12% NuPAGE gels (Invitrogen) and transferred using a semi-dry transfer system onto PVDF membranes (Millipore). Non-specific binding sites were blocked with 10% milk TBS-T solution for 1 h at room temperature, then probed overnight at 4 °C with the respective primary antibodies: anti-SAMHD1 (Abcam), anti-SOX11 (Sigma), or anti-Cyclin D1 1:1000 in 5% milk or 5% BSA in TBS-T. Membranes were then washed in TBS-T and probed with secondary antibodies (HRP-conjugated anti-rabbit or anti-mouse; GE Healthcare). Blots were developed using Supersignal West Pico (Pierce) and visualized using LiCor machine. For re-probing of membranes with anti-actin (Sigma) or anti-GAPDH (Cell Signaling) (1:5000 in 5% milk in TBS-T, Sigma), HRP was blocked using the SG substrate kit (Vector Labs). Analysis was done using Fiji-ImageJ software.