SDF-1 induced CXCR4 downregulation is TSG101-dependent. (A) COS-1 cells co-transfected with HA-tagged CXCR4 and either GFP, TSG101-GFP, or siRNA directed against TSG101, as indicated, were incubated with an anti-HA antibody for 1 hour on ice. Cells were then either fixed and stained with a secondary antibody to detect cell surface CXCR4 (first row), or incubated in DMEM/10% FBS without SDF (second row) or with 100 nM SDF (third and fourth rows) for 3 hours at 37°C prior to fixation, permeabilization and secondary-antibody staining. The cells were then analysed for GFP fluorescence (green), or CXCR4 expression (red). Blue represents nuclear staining. Scale bars = 10 μm. (B) Immunoblot of COS-1 cells co-transfected with HA-tagged CXCR4 and siRNA directed against either TSG101 or LacZ. Top panel, TSG101 Western blot; bottom panel, actin Western blot.