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. Author manuscript; available in PMC: 2017 Nov 27.
Published in final edited form as: Cell Rep. 2017 Mar 14;18(11):2600–2607. doi: 10.1016/j.celrep.2017.02.051

Figure 3. TREX1 DNase Activity Is Not Affected by Phosphorylation.

Figure 3

(A) TREX1 DNase activity was measured by a real-time fluorescent assay using a 30-nt-long ssDNA as a substrate. HeLa cells and HeLa cells stably expressing TREX1-V5 were grown asynchronously (AS) or arrested in mitosis with nocodazole (M). Cell extracts were immunoprecipitated with anti-V5 agarose beads. After the assay is completed, aliquots of the reaction mixtures were examined by immunoblot of Phos-Tag PAGE to confirm status of TREX1 phosphorylation (right).

(B) TREX1-V5 WT, S78A/261A, and S78D/261D mutants were expressed in 293T cells and immunoprecipitated using anti-V5 agarose beads. DNase activity was measured as in (A). After the assay ended, aliquots of the reaction mixtures were examined by immunoblots with regular SDS-PAGE (bottom).

(C) TREX1-V5 WT and a S166A/167A mutant were expressed in 293T cells and immunoprecipitated using anti-V5 agarose beads. DNase activity was measured as in (A). After the assay ended, aliquots of the reaction mixtures were examined by immunoblots with regular SDS-PAGE (bottom). Data are representative of at least three independent experiments. Error bars, SD.