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. Author manuscript; available in PMC: 2017 Dec 14.
Published in final edited form as: Cell Host Microbe. 2016 Nov 17;20(6):770–784. doi: 10.1016/j.chom.2016.10.011

Figure 4. UL37 deamidates RIG-I in cells and in vitro.

Figure 4

(A) HEK293/Flag-RIG-I cells were transfected with an empty or UL37-containing plasmid. Whole cell lysates (WCLs) were analyzed by two-dimensional gel electrophoresis and immunoblotting with the indicated antibodies.

(B and C) HEK293/Flag-RIG-I cells were transfected with a UL37-expressing plasmid or infected with HSV-1 (MOI=1) for 12 h. RIG-I was purified and analyzed by tandem mass spectrometry. Two peptides containing deamidated asparagines were identified. D495 and D549 (in red) were shown (B). Deamidated peptides were quantitatively determined by tandem mass spectrometry analysis and data represents one of two independent experiments (C).

(D) HEK293/Flag-RIG-I or HEK293/Flag-RIG-I-DD cells were transfected with an empty or UL37-containing plasmid. WCLs were analyzed by two-dimensional gel electrophoresis and immunoblotting.

(E) GST-RIG-I and UL37 were purified from transfected 293T cells and E.coli, respectively, and analyzed by silver staining (right panels). Deamidation reaction was analyzed by two-dimensional gel electrophoresis and immunoblotting with anti-RIG-I antibody.

See also Figure S4.