Figure 5
From: Identification and characterization of Iporin as a novel interaction partner for rab1
Cellular distribution of Iporin. (Aa) To test specificity of the polyclonal anti Iporin antibody Aip519, HeLa cells were transiently transfected with HA-tagged Iporin. After lysis, cell extracts were blotted and incubated with the Aip519 and the monoclonal 16B12 anti HA antibody, respectively. The Western blot displays a signal of about 220 kDa for both antibodies. (Ab) Cytosol/membrane fractionation with extracts from transiently transfected HeLa cells. Recombinant HA-Iporin was detected with anti HA (16B12) antibody. Fractionation of full-length HA-Iporin and HA-Iporin ΔN847 revealed that most of the recombinant proteins were present in the membrane fraction (P100) with small amounts in the cytosolic (S100) fraction. In contrast, the transiently expressed HA-Iporin ΔC853 was present in a high concentration in the cytosolic (S100) fraction. (Ac) Comparison between untransfected and HA-rab1bQ67L transfected HeLa cells. Blots were analyzed using the Aip519 (Iporin), anti-GM130 (GM130), anti-p115 (p115), anti-calnexin and 1E7 (anti-rab1b) antibodies. (B) Immunofluorescence staining of recombinant HA-Iporin expressed in HeLa cells shows a cytosolic distribution with a perinuclear enrichment in spotted structures. Bar 10 μm.