EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination
Figure 2
EphA2 is activated and associated with entry receptors early during KSHV infection.
(A) Serum-starved (8 h) HFF cells were either mock infected (UI) or infected with KSHV (30 DNA copies/cell) for the indicated time periods and subjected to Western blot analysis for phospho-EphA2 (Y594) (pEphA2). The blot was stripped and reprobed for total EphA2 and tubulin was used as loading control. (B) Serum starved HFF cells were either mock infected or infected with KSHV (30 DNA copies/cell) for 5 and 10 min and processed for immunofluorescence analysis using rabbit anti-EphA2 and mouse monoclonal anti-KSHV gpK8.1A antibodies for overnight at 4°C followed by staining with anti-rabbit Alexa 488 and anti-mouse Alexa 594 secondary antibodies. Representative 2D convoluted images are shown. The white boxes within the merged panels are shown as enlarged pictures and the white arrows represent colocalization of the indicated molecules. (C) Serum-starved HFF cells were left uninfected or infected with KSHV for the indicated periods of time and immunoprecipitated with anti-α3β1, αVβ3 or αVβ5 antibodies and analyzed for EphA2 by Western blot (first, third and fifth panels). These blots were stripped and reprobed for total β1, β3 and β5 integrin subunits, respectively (second, fourth and sixth panels). For negative control, cell lysates from uninfected or KSHV infected HFF cells were immunoprecipitated with anti-β6 antibody and analyzed for EphA2 by western blotting (seventh panel). The blot was stripped and reprobed with total β6 antibodies (eighth panel). Whole cell lysates were subjected to western blot analysis for input expression of EphA2 (ninth panel) and β6 (tenth panel) and β-actin was used as loading control (eleventh panel). (D) Serum starved HFF cells were either left uninfected or infected with KSHV (30 DNA copies/cell) for 5, 10 and 30 min, washed and processed for immunofluorescence analysis. Cells were permeabilized, blocked with blocking reagents and incubated with (D1) rabbit anti-EphA2 and mouse anti-αVβ3 antibodies or (D2) rabbit anti-EphA2 and mouse anti-αVβ5 antibodies for 2 h at room temperature followed by staining with anti-rabbit Alexa fluor 488 or anti-mouse Alexa fluor 594. Representative 2D deconvoluted images are shown. The enlarged pictures represent boxed regions within the merged panels and the white arrows represent colocalization of the indicated molecules.