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Figure 4 | Virology Journal

Figure 4

From: Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch

Figure 4

UL56 interacts with Itch and changes the subcellular localization of Itch. Co-immunoprecipitation assay on HSV-2-infected cells (A) or stably UL56-expressing cells (B). (A)Vero cells were treated with chloroquine (CQ) for 12 h and subsequently mock-infected or infected with wild-type HSV-2 (186). Whole cell lysates (WCL) were immunoprecipitated (IP) with an anti-Itch antibody at 9 hpi. (B) WCL from Vero-GFP or Vero-GFP-UL56 cells treated with CQ for 24 h and immunoprecipitated with an anti-Itch antibody. UL56 was detected in the Itch-immunoprecipitates in HSV-2-infected cells (A) and stably UL56-expressing cells (B). β-actin was used as a loading control. Confocal immunofluorescence analysis of the subcellular localizations of Myc-Itch and UL56 in HSV-2-infected cells (C) or co-expressing cells (D). (C) HEp-2 cells were transfected with plasmids encoding Myc-Itch and subsequently infected with wild-type HSV-2. The Myc-Itch (red) showed the altered subcellular distribution with the reduced signal intensity after 6 hpi, when UL56 (green) became detectable. Myc-Itch colocalized with UL56 in the vesicular pattern. (D) HEp-2 cells were transfected with plasmids encoding UL56 (pcDNA-UL56) and/or pCMV-Myc-Itch. In co-expressing cells (bottom panels), Myc-Itch changed its distribution and colocalized with UL56 in the vesicular pattern. The Myc signal was reduced in co-expressing cells. Scale bars, 10 μm.

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