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. Author manuscript; available in PMC: 2018 Jul 31.
Published in final edited form as: Cell Rep. 2018 Jul 24;24(4):815–823. doi: 10.1016/j.celrep.2018.06.090

Figure 4. Effects of Cysteine Mutations on the Restriction Activity, dNTP Depletion, and T592 Dephosphorylation.

Figure 4

(A) PMA-treated U937 cells stably expressing the indicated SAMHD1 variants were challenged with increasing amounts of HIV-1-GFP. Infection was determined by measuring the percentage of GFP-positive cells using flow cytometry. n = 3, where n denotes the number of independent viral challenges with all mutants, but only one representative set of challenges is shown. The same mutant-dependent trends were observed in all three replicate experiments.

(B) Total cellular levels of dATP and deoxyguanosine triphosphate (dGTP) were measured in PMA-treated U937 cells stably expressing SAMHD1 variants using a primer extension assay, as described in Experimental Procedures. n = 3, where n represents the number of independent dNTP concentration measurements using the primer extension assay. Error bars denote SD.

(C and D) Cysteine mutations do not affect dephosphorylation of T592 following PMA treatment of U937 cells stably expressing WT and mutant SAMHD1 variants. (C) T592 is phosphorylated in U937 cells expressing SAMHD1 and not treated with PMA in all SAMHD1 variants tested. SAMHD1 expression levels are low in U937 cells before PMA treatment, so the phosphorylation status was evaluated on immunoprecipitated SAMHD1 (see Experimental Procedures).

(D) Following PMA treatment, dephosphorylation of T592 is evident in the WB analysis of the crude cell extracts. Cysteine mutations have no effect on T592 dephosphorylation. Dephosphorylation of the endogenous SAMHD1 in PMA-treated and untreated THP-1 cells is shown as control in all gels.

See also Figure S4.