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Figure 4 | Retrovirology

Figure 4

From: Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Figure 4

SAMHD1 expression and function are not affected by CCL2 neutralization in MDM. (A) MDM were treated with anti-CCL2 or control Ab (2.5 μg/ml) for 20 h. Total RNA was then extracted, retrotranscribed, amplified and the 2ΔΔCt values for SAMHD1 transcripts were calculated as described in Methods. (B) MDM were treated as in A and SAMHD1 protein expression and phosphorylation in whole cell extracts were detected by western blot. Actin was used as house-keeping gel loading control. (C) MDM were treated as in A and then infected with HIV-1BaL (3000 TCID50 per well) as described in Methods. Cells were lysed 14 days after infection and SAMHD1 protein expression in whole cell extracts was detected by western blot. Actin was used as house-keeping control. (D) MDM were treated and infected as in C either in the absence or in the presence of dNTPs (1.5 mM) as described in Methods. After 14 days, cells were recovered and HIV-1 Gag expression was evaluated by flow cytometry. (E) MDM were treated as in A and then challenged with LV/Vpx expressing GFP (2 × 106 TU per 106 cells). The proportion of GFP+ cells was determined 3 days post-infection by flow cytometry. The effect of Vpx on SAMHD1 protein expression is shown in the inset. In A and E, data represent mean values (+SE) of the results obtained with 4 different donors. In B, C and D, the result from a single representative experiment out of 4 independently performed is shown.

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