PC1/3 ER-retained mutants induce ER-stress in Min6 cells. Min6 cells were transfected with cDNAs encoding either PC1/3 ER-retained mutants, or the PC1/3 N309K inactive mutant (control). PC1/3 mutant-ir was detected using M2 anti-FLAG. A, BiP-ir was detected using a polyclonal rabbit antibody (Abcam). B, Xbp-1-ir was detected using a polyclonal rabbit antibody against Xbp-1. Nuclei were stained with DAPI. Immunofluorescence images show that PC1/3 N309K exhibits a predominantly perinuclear pattern, whereas the PC1/3 ER-retained mutants show a reticular pattern. A1, BiP-ir exhibits a reticular pattern and colocalizes with ER-retained PC1/3 mutants. B1, Xbp-1-ir exhibits a nuclear pattern and colocalizes with DAPI. Expression was analyzed as a percentage of PC1/3-transfected cells exhibiting BiP or Xbp-1 expression. Data were analyzed using a one-way ANOVA followed by a post hoc Bonferroni's test (***, P < .001; **, P < .01; *, P < .05); n = 20 from 3 different experiments. C, Western blotting shows increased BiP expression in Min6 cell lysate samples transfected with PC1/3 ER-retained mutants.