shRNA-mediated downregulation of SNAP23 disrupts β
integrin trafficking into the recycling endosome. Hela cells were transiently transfected for 72 h with GFP + PLOK-1 vectror [A-D] or GFP + vector containing SNAP23-targeting shRNA [E-H]. Cells were labeled with anti-β1 integrin antibody in serum free media for 1 h to allow internalization of integrin-antibody complexes. Cells were then washed in 0.2 M glycine, pH 2.5, fixed, and stained for the intracellular integrin-antibody complexes [B, F] and Rab11 [C, G]. Overlays of the Rab11 and β1 integrin staining are shown in D and H. Arrows indicate conventional colocalization of β1 integrin in a central Rab11-containing compartment. Images are 3 D reconstructions of a z-series. Scale bar represents 10 μm.