FIGURE 2:

cTAGE5 mutants lacking Sec12-binding ability fail to recruit Sec12 to the ER exit sites. (A) HSC-1 cells were transfected with cTAGE5 siRNA and cultured for 24 h. cTAGE5-FLAG wild-type and mutant constructs were transfected, and the cells were cultured for another 24 h, fixed, and stained with Sec12 (clone 6B3), FLAG, and Sec16 antibodies. Scale bar, 10 μm. Typically, >90% of the cells showed efficient reduction of cTAGE5 expression by siRNA transfection (cells with asterisks). Less than 10% of the cells were transfected with FLAG-tagged constructs. Cells with asterisks indicate that the cells were efficiently depleted for cTAGE5, but FLAG-tagged constructs were not transfected. (B) Quantification of Sec12 immunofluorescence intensity at ER exit sites in A. A.U., arbitrary units. Fifty ER exit sites from 10 cells, n = 50 (analysis of variance). Error bars represent mean ± SEM; **p < 0.001 compared with wild-type expression; n.s., p > 0.05 compared with wild-type expression. The data shown are from a single representative experiment out of three repeats.