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. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: Traffic. 2010 May;11(5):660–674. doi: 10.1111/j.1600-0854.2010.1045.x

Figure 2.

Figure 2

Post-endocytic sorting of US28 is mediated by GASP-1. A) The dominant negative cGASP-1 binds to GST-fusion proteins containing the CT of US28, the DOP, but not the GST-protein (C), beads (B) alone or the MOP. The corresponding input protein levels are shown below the radiographs. Blots are representative of at least three independent experiments. B) cGASP-1 competes with GASP-1 for the binding site on the US28-CT. MBP-US28 was incubated with increasing concentrations of purified cGASP-1 in the presence of 35S-Met-labelled recombinant GASP-1. MBP-protein was used as control (C). Molecular weight marker (S). The corresponding input protein level is shown below the radiograph. C) US28 is sorted to lysosomes. HEK293 cells transiently transfected with Flag-US28 were ‘fed’ antibody to the extracellular Flag-tag for 45 min, then fixed and permeabilized. Receptors (green) were analysed for colocalization with the lysosomal markers LAMP1 and LAMP2 (red), merge: yellow. D) cGASP-1 alters the localization of US28. Antibody feeding experiments in HEK293 cells stably expressing GFP-cGASP-1 (see insert) and transiently expressing Flag-US28 revealed a redistribution of the receptor to vesicles close to the cell surface (see arrows). Green: Flag-US28, red: LAMP1/2, merge: yellow. E) Knock-down of endogenous GASP-1 in HEK293 cells using a shRNA-lentivirus. HEK293 cells were infected with either shGASP-1 or shScr virus. Forty-eight hours post-infection, cells were lysed, separated by SDS/PAGE and immunoblotted for GASP-1. β-actin staining served as loading control (lower panel). GASP-2 levels remained unchanged in both shScr and shGASP-1 infected cells (see 130-kDa band). F) EGF receptor degradation is not impaired by shGASP-1 knock-down. HEK293 cells infected with shScr or shGASP-1 virus were transiently transfected with Flag-US28 and either left untreated or incubated with EGF (5 μm) for 3 h. Lysates were analysed by SDS/PAGE and immunoblotted for endogenously expressed EGF receptor (first panel, EGFR), GASP-1 and GASP-2, US28 and β-actin (lower panels). G,H) shRNA knock-down of GASP-1 prevents lysosomal targeting of US28. HEK293 cells were infected with either shScr virus (G) or shGASP-1 virus (H) and transfected with Flag-US28. Antibody feeding experiments showed that US28 colocalizes with LAMP1 and LAMP2 in cells infected with scrambled virus (G), whereas the receptor is redistributed to vesicles close to the cell surface in cells infected with shGASP-1 (H, see arrows). Green: Flag-US28, red: LAMP1/2, merge: yellow, insert: EGFP-shRNA-virus. Scale bars = 10 μm.