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. 2012 Aug 21;31(20):3976–3990. doi: 10.1038/emboj.2012.235

Figure 2.

Figure 2

Immunoisolation of the TGN46 containing transport carriers. (A) The anti-TGN46 antibody recognizes the cytoplasmic tail of TGN46. HeLa cells were permeabilized with digitonin, fixed with paraformaldehyde, incubated with or without Triton X-100 (TX-100), and then visualized with affinity purified anti-TGN46 and anti-mannosidase II antibodies. Bar, 10 μm. (B) HeLa cells were transfected with control (Cont) siRNA or siRNA oligos specific for TGN46. This procedure was repeated after 24 h. Seventy-two hours after the first transfection the cells were lysed and the lysates were western blotted with the anti-TGN46 antibody. (C) Permeabilized HeLa cells were incubated with an ATP regenerating system and rat liver cytosol at 32°C. The low speed supernatant of the reaction mixture was incubated with or without anti-TGN46 antibody and the immunoprecipitates were western blotted with anti-TGN46 antibody. An aliquot of the low speed supernatant was centrifuged at high speed (100 000 g for 1 h) and the pellet was analysed (Total transport carriers (TC)). The single and double stars denote immunoglobulin heavy chain and a non-specific polypeptide, respectively. (D) Permeabilized HeLa cells were incubated with an ATP regenerating system and rat liver cytosol at 4 or 32°C. Immunoprecipitates were western blotted with indicated antibodies. (E) Low speed supernatant of reaction mixture was prepared from HeLa-ssHRP cells and subjected to the immunoprecipitation in the presence or absence of TX-100. The HRP activity in immunoprecipitated materials was quantified by chemiluminescence. The average values of two experiments are shown (mean±s.d.). (F) Immunoisolated TGN46 containing membranes were released from the magnetic beads by incubation with 100 mM sodium carbonate (pH 11.5) and collected by high speed centrifugation (100 000 g for 1 h). The membranes were western blotted with indicated antibodies: single and double stars denote immunoglobulin heavy chain and a non-specific polypeptide, respectively. Figure source data can be found with the Supplementary data.