EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination
Figure 11
c-Cbl regulates clathrin mediated productive entry of KSHV without affecting its association with EphA2.
(A and B) Serum starved, control si-RNA or c-Cbl-si-RNA transfected, HFF cells either uninfected or infected with KSHV for 5 min were subjected to immunofluorescence assay using anti-rabbit KSHV gB and mouse anti-clathrin antibodies (A1) or anti-rabbit EphA2 and anti-mouse KSHV gpK8.1 antibodies (B1), respectively, overnight at 4°C followed by staining with either anti-rabbit Alexa 594 and anti-mouse Alexa 488 (A1) or vice versa (B1). Representative 2D convoluted images are shown. The enlarged pictures represent boxed regions within the merged panels and the white arrows represent colocalization of the indicated molecules. (A2 and B2) Quantification of KSHV colocalized with clathrin or EphA2 was performed by counting three different fields having atleast 10 cells each and error bars represent mean ± SD, ** represents p<0.01. (C) Control or c-Cbl-si-RNA-transfected HFF cells were incubated with medium containing LysoTracker green along with or without KSHV (30 DNA copies/cell) for 30 min at 37°C and were processed for immunofluorescence assay using anti-gB antibody followed by Alexa 594 anti-rabbit secondary antibody. 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.