Fig 5

Fig 5

(A, B) COS-7 cells were first transfected with an expression vector for human APOB48 (5 μg), distributed equally in 6-well plates, and subsequently transfected with plasmids expressing either wild-type zebrafish mttp-FLAG, mttp^stl-FLAG, mttp^c655-FLAG, or empty vector (pcDNA3) (3 μg). After 72 h, APOB48 was measured via ELISA in media (A) or in the cell (B). Data are representative of 7 independent experiments (each data point is the mean of three technical replicates), mean +/- SD, One-Way ANOVA with Bonferroni post-hoc tests, * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Representative immunofluorescent staining using anti-FLAG (red) and anti-Calnexin (green) antibodies in COS-7 cells expressing wild-type or mutated mttp-FLAG constructs; scale = 25 μm. The percentage of cells expressing the FLAG-tagged proteins was similar among all groups (Mtp-FLAG 37%, stl-FLAG 31%, c655-FLAG 41% transfection efficiency). (D) Zebrafish Mtp-FLAG proteins (WT, stl and c655) were immunoprecipitated from COS-7 cell lysate (400 μg) using the M2 flag antibody and immunoblots were probed for both FLAG and PDI (Representative of 2 experiments). For input, 15 μg of cell lysate was used. (E) COS-7 cells were transfected with plasmids expressing pcDNA3, wild-type zebrafish mttp-FLAG, or mutant mttp-FLAG constructs. Cells were lysed and 60 μg of protein was used to measure the % TG transfer of nitrobenzoxadiazole (NBD)-labeled triolein from donor to acceptor vesicles after 45 min; n = 3 (each n is the mean of three technical replicates from independent experiments), mean +/- SD, One-way ANOVA with Bonferroni post-hoc tests, *** p < 0.001. (F) Wild-type and mutant Mtp proteins were purified using anti-FLAG antibodies and used to measure the % transfer of NBD-labeled phosphoethanolamine triethylammonium from donor to acceptor vesicles after 180 min; n = 3 (each n is the mean of three technical replicates from independent experiments), mean +/- SD, randomized block ANOVA with Bonferroni post-hoc tests, *** p < 0.001.