Fig. 5

FAM91A1 residues interacting with TBC1D23 are required for endosome-to-Golgi trafficking of KIAA0319L. (A) Subcellular location of KIAA0319L in HeLa cells. The FAM91A1 KO cells were transfected with GFP, GFP-tagged FAM91A1 WT, R61A, KDAA, or D198R respectively. Cells were then incubated with antibodies against KIAA0319L (gray) and golgin-97 (red) (Scale bar, 10 μm). (B) Colocalization analysis between KIAA0319L and golgin-97 in A. Each dot represents Pearson’s correlation coefficients from one cell. Data are presented as mean ± SD, and P values were calculated using one-way ANOVA and Tukey’s multiple comparisons tests. Ns: not significant, *P < 0.05, 0.001 < ****P < 0.0001; the figure is representative of n = 3 independent experiments with similar results. (C) Immunoblot of whole-cell extracts showing that knockout of FAM91A1 decreased the total protein level of KIAA0319L in HEK293T cells. Transient expression of WT, but not these mutants or GFP vector, rescued the reduction. (D) The relative abundance of KIAA0319L compared to GAPDH was quantified in (E) and compared to the WT group. Data are presented as mean ± SD, and P values were calculated using one-way ANOVA and Tukey’s multiple comparisons tests. Ns: not significant, *P < 0.05, 0.05 < **P < 0.001; (E) Immunoblot of entire zebrafish tissue extracts showing that injection of tbc1d23 MO or fam91a1 MO decreased the protein level of KIAA0319L.