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. 2017 Dec 4;216(12):4153–4164. doi: 10.1083/jcb.201705027

Figure 5.

Figure 5.

cTAGE5 depletion affects the Sec22b-SNARE and Sec22b-COPII interaction. (A) MIN6 cell line stably expressing scramble shRNA (siControl) or Sec22b shRNA (siSec22b) was infected with mPro-CpepSfGFP expressing lentivirus. 48 h later, cells were lysed and probed for indicated proteins. Percentage of CpepSfGFP to total mPro-CpepSfGFP is quantitated in the right panel. (B) The interaction of endogenous Sec22b with Sec31a, cTAGE5, Sec23a, and Syntaxin5 in MIN6 cells was observed by coIP with Sec22b antibody. However, the interaction with Sec31a and Syntaxin 5 was much weaker in cTAGE5 knockdown cells. Quantitation of protein levels is shown in the right panel. All data are means ± SEM (Student’s t test) from three independent experiments. ***, P < 0.001; ns, not significant. (C) Constructs expressing Sec31a, Sec22b, and cTAGE5 were transfected alone or in combination into 293 cells; 24 h later, cell lysates were immunoprecipitated with Flag agarose beads and the immune complexes were detected with indicated antibodies. *, IgG band. (D) Control and cTAGE5 knockdown MIN6 cells stained for Sec22b and Sec31a. Bar, 10 µm. (E) MIN6 cells were incubated with BFA (5 µg/ml) for 1 h at 37°C, washed with PBS, and then fixed and stained with Sec22b, cTAGE5, or Sec31a antibodies. Bar, 10 µm. Ctrl, control.