Ab67820

Human Embryonic Kidney (HEK) 293T cells were cultured in DMEM (Invitrogen). The human monocytic U937 and THP1 cell lines were grown in RPMI (Invitrogen). Media were supplemented with 10% fetal calf serum (Invitrogen) and penicillin/streptomycin (100 U/mL). U937 and THP1 cell lines were differentiated by treatment with phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) (300 ng/mL, 24 h). All cell lines were tested mycoplasma-free (Mycoplasmacheck, GATC Biotech). Buffy coats from human healthy donors were obtained from the “Etablissement Français du Sang”. Monocytes were isolated using a CD14+ selection kit (Miltenyi Biotech) and cultured 12 days in DMEM supplemented with 10% Human Serum (inactivated) to generate MDMs. Antibodies used are the following: sheep anti-SUMO1 (Enzo, 1:1000 for WB), rabbit anti-SUMO1 (ab32058, 1:1000 for PLA), rabbit anti-SUMO2/3 (ab3742, 1:1000 for WB, 1:3500 for PLA), mouse anti-SUMO2/3 (ab81371, 1:6000 for PLA), mouse anti-SAMHD1 (ab67820, 1:1000 for WB, 1:5000 for IP, 1:2500 for PLA), rabbit anti-SAMHD1 (ab177462, 1:6000 for PLA), rabbit anti-pT592-SAMHD1 (Cell Signaling #89930, 1:1000 for WB), rabbit anti-actin (Sigma-Aldrich, AA20-33, 1:2500 for WB), anti-HA HRP (Roche Clone 3F10, 1:2500 for WB), Alexa Fluor 488 anti-mouse, Alexa Fluor 594 anti-mouse (INVITROGEN, 1:800 for IF).

For analysis of the levels of expression of total and phosphorylated SAMHD1 in siRNA-transfected iDCs or MDMs, as well as in MDM-derived MGCs, cells (10⁶) were lysed in 100 μl of reducing Laemmli sample buffer (2×) supplemented with PhosSTOP (phosphatase inhibitor cocktail tablets; Sigma-Aldrich) and cOmplete (EDTA-free protease inhibitor cocktail; Sigma-Aldrich) and then boiled at 96°C for 10 min. Cell lysates were then resolved by SDS-PAGE as described previously (64 (link)) and analyzed by Western blotting using anti-SAMHD1 (Abcam; ab67820), anti-phospho-SAMHD1 (Cell Signaling; catalog no. 89930), anti-HIV-1 p24 (Abcam; ab9071), and anti-β-actin (Sigma-Aldrich; clone AC-15) antibodies.