FIGURE 3:

Sar1 coexpression with cTAGE5 mutant recovers collagen VII secretion from the ER. HSC-1 cells were treated with control or cTAGE5 siRNA and cultured for 24 h. For cTAGE5 siRNA-treated cells, cTAGE5-FLAG wild type or mutants (A) or cTAGE5-FLAG constructs together with HA-Sar1a constructs (B) were transfected and further cultured for 24 h. The cells were fixed and stained with collagen VII and FLAG (A) or collagen VII, FLAG, and HA antibodies (B). Collagen VII immunofluorescence signal per cell (A.U., arbitrary units) were quantified in each cell category as described later. The cells positively stained with FLAG or HA antibodies were categorized as the constructs expressed, and the surrounding unstained cells were categorized as nontransfected counterparts. Within each well, cells transfected with constructs are labeled as +, and nontransfected cells are labeled as –. Analysis of variance. Error bars represent mean ± SEM; **p < 0.001; *p < 0.05; n.s., p > 0.05. The data shown are from a single representative experiments out of three repeats. (A) Cells treated with control siRNA (n = 78); cells treated with cTAGE5 siRNA and wild type– (n = 140); wild type+ (n = 49); 60-300aaT1– (n = 111); 60-300aaT1+ (n = 49); S68A R69A– (n = 131); S68A R69A+ (n = 50); E75A K76A– (n = 114); E75A K76A+ (n = 48); and K89A– (n = 167); K89A+ (n = 51). (B) Cells treated with control siRNA (n = 75); cells treated with cTAGE5 siRNA and HA-Sar1aWT– (n = 62); HA-Sar1aWT+ (n = 12); HA-Sar1aH79G– (n = 135); HA-Sar1aH79G+ (n = 37); E75AK76A–, Sar1aWT– (n = 358); E75AK76A+, Sar1aWT– (n = 74); E75AK76A+, Sar1aWT+ (n = 54); E75AK76A–, Sar1aH79G– (n = 272); E75AK76A+, Sar1aH79G– (n = 67); and E75AK76A+, Sar1aH79G+ (n = 54).