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Fig. 5 | Cell & Bioscience

Fig. 5

From: PIAS1 potentiates the anti-EBV activity of SAMHD1 through SUMOylation

Fig. 5

Phosphorylation enhances SAMHD1 SUMOylation and SUMOylated SAMHD1 can be phosphorylated by viral protein kinases. A Schematic representation of SAMHD1 phosphorylation and subsequent SUMOylation analysis. Step 1: Recombinant SAMHD1 was phosphorylated by EBV protein kinase BGLF4 in vitro. Non-phosphorylated SAMHD1 was similarly processed by incubating with Buffer control. Step 2: The phosphorylated and non-phosphorylated SAMHD1 were used to perform in vitro SUMOylation reaction using E1, E2, SUMO2 and PIAS1 or buffer control. Step 3: The reaction was terminated by adding 2 × SDS sample loading buffer and the samples were analyzed by WB. B Phosphorylated and non-phosphorylated SAMHD1 generated in (A) Step 1 were incubated with buffer control or with SUMOylation machinery. The SUMOylation and phosphorylation of SAMHD1 were analyzed by WB using anti-SAMHD1 and anti-p-SAMHD1 (T592) antibodies as indicated. Arrows denote mono-or di-SUMOylated SAMHD1. C Schematic representation of SAMHD1 SUMOylation and subsequent phosphorylation analysis. Step 1: Recombinant SAMHD1 was SUMOylated by E1, E2, SUMO2 and PIAS1 in vitro. Non-SUMOylated SAMHD1 was similarly processed by incubating with buffer control. Step 2: The SUMOylated and non-SUMOylated SAMHD1 were subjected to in vitro kinase reaction using viral protein kinases (EBV BGLF4 or HCMV UL97). D WB analysis showing that SUMOylated SAMHD1 was generated from (C) Step 1. Arrows and bracket denote mono- and poly-SUMOylated SAMHD1, respectively. E SUMOylated and non-SUMOylated SAMHD1 generated in (C) Step 1 were incubated with buffer control or with EBV BGLF4 and HCMV UL97 in step 2. As controls, SAMHD1-RRR mutant samples from Fig. 4B (lanes 7 and 8) were also incubated with EBV BGLF4 and HCMV UL97. The phosphorylation status was analyzed by WB using anti-p-SAMHD1 (T592) antibodies. Arrows denote mono- or di-SUMOylated SAMHD1

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