Fig 1: Alteration of the ER entry pathway of Prion and LY6K affects their N-glycosylation and GPI structure.A, HEK293 WT, STT3A-KO (clone #4), and STT3B-KO (clone #1) cells were transfected with FLAG-tagged Prion, LY6K, and CD59. Cell lysates treated with or without PNGase F (PNG-F) were Western blotted for Prion, FLAG, STT3A, TUSC3, and GAPDH. Lower panel of FLAG (CD59) shows the long-exposed image. * indicates nonspecific bands. B, HEK293 WT, STT3A-KO, and STT3B-KO cells were transfected with chimeric Prion and LY6K constructs. Cell lysates were Western blotted for Prion, FLAG, and GAPDH. C, the surface expression of CD59 and Prion treated with or without PI-PLC is shown. Cells were stained with anti-CD59 or anti-Prion antibodies. D, the surface expression of chimeric Prion in WT cells treated with or without PI-PLC is shown. Cells were stained with anti-Prion antibody. The graph shows the quantification of relative fluorescence intensity of chimeric Prion constructs treated with or without PI-PLC in WT cells. Error bars represent SD (n = 3). Statistical analysis was performed by one-way ANOVA with post hoc Dunnett test. * p < 0.05, ** p < 0.005. ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol.