Both E449 and E452 of Mlph are essential for the perinuclear distribution of melanosomes induced
by Mlph-ΔRBD overexpression. Melan-a melanocytes were transfected to express EGFP-Mlph-ΔRBD (WT, E449A, or E452A) or EGFP. The distribution of melanosomes in the transfected melanocytes was imaged and the number of melanocytes with perinuclear distribution of melanosomes was counted. (A) Typical images of melanocytes expressing EGFP alone, EGFP-Mlph-ΔRBD WT, E449A, or E452A. Zooms are x 3. Cells are outlined with red dashed lines. Scale bars = 10 µm. The transfection efficiences were 11% for EGFP (n=275), 9.2% for EGFP-Mlph-ΔRBD WT (n=194), 11.2% for E449A (n=208), and 11.8% for E452A (n=267). n represents cell number. (B) The percentage of melanocytes exhibiting perinuclear melanosome aggregation among the transfected melanocytes. Results are presented as the mean ± SD of three independent experiments with one-way ANOVA with post-hoc Bonferroni test. Each point represents the percentage of perinuclear distribution of EGFP fusion proteins expressing cells in each fields. ****ρ<0.0001. (C) Western blot of the lysates of melan-a transfected with EGFP-Mlph-ΔRBD WT, E449A, or E452A. The expressed EGFP fusion proteins were probed with the antibody against EGFP. The relative band densities of EGFP-Mlph-ΔRBD WT, E449A, and E452A were 1, 1.29, and 1.25, respectively. Correction of the transfection efficiencies of those three constructs (Figure 6A, Figure 6-figure supplement) gave rise to the relative protein expression levels of each transfected cells as 1, 1.05, and 0.97 for EGFP-Mlph-ΔRBD WT, E449A, and E452A, respectively.
Figure 6-Source data 1 Original and statistical data for Figure 6 B.
Figure 6-Source data 2 Original and uncropped blots for Figure 6 C.