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Fig. 6. Syndapin II is bound to Golgi-enriched membranes of HepG2 cells and can be localized to the Golgi complex of COS-7 cells by immunofluorescence analysis. (A) After incubation of HepG2 cells at 37°C or at 20°C (Golgi exit block), syndapin II is detected in light membranes (mem.) and in Golgi-enriched membranes but not in heavy membranes sedimenting through 1.2 M sucrose. 30 µg membrane proteins were separated by SDS-PAGE, blotted and analyzed with anti-syndapin II antibody 3685. (B-J) Syndapin II (Sdp II) co-localizes with the Golgi marker syntaxin 6 in both untransfected COS-7 cells and in cells transfected with syndapin II-l, as seen well in the merged images (D-J; syndapin II in green, syntaxin 6 in red). Syndapin II was detected by the syndapin II-specific antibody P339 (B,E,H) and syntaxin 6 was detected with a monoclonal anti-syntaxin 6 antibody (C,F,I). Similar to endogenous syndapin II, overexpressed Xpress-tagged syndapin II-l also shows a perinuclear co-localization with syntaxin 6 (B-D). The two cells expressing Xpress-tagged syndapin II-l shown in B are marked by asterisks. Inserts show a shorter exposure of perinuclear region of the upper transfected cell. (E-G) High magnifications show that endogenous syndapin II (E) co-localizes very well with syntaxin 6 (F). (H-M) After incubation of COS-7 cells with brefeldin A to disrupt the TGN, syntaxin 6 immunostaining was found either to be collapsed into small perinuclear dots or to be dispersed (I,L). The immunostaining of endogenous (H) and overexpressed syndapin II (K) was mainly dispersed and showed no co-localization with the perinuclear syntaxin 6 accumulations formed upon BFA treatment (J,M). Bars, 10 µm.
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