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. 2022 Nov 16;23(22):14144. doi: 10.3390/ijms232214144

Figure 5.

Figure 5

Black-pigmented melanosomes formed normally in Rab32/Rab38-KO B16-F1 cells. (A,B) Domain organization of the mouse Rab32 and Rab38 proteins and genomic mutations in Rab32/38-KO cells. The Cas9 target and PAM sequences are highlighted in magenta and green, respectively. Genomic PCR products containing the target sites were subcloned into the pGEM-T Easy vector. Then, nine colonies for Rab32 and 15 colonies for Rab38 (denominators of the fractions in parentheses) were picked up at random and their inserts were sequenced. The numerators of the fractions in the parentheses are the numbers of colonies obtained by sequencing the subcloned PCR products. (C) Expression of Rab32, Rab38, Tyr, and Tyrp1 in WT cells and Rab32/38-KO B16-F1 cells as determined by immunoblotting with the antibodies indicated. (D) Quantification of the melanin content (i.e., optical density at 490 nm) in WT cells, Tyr-KO cells, and Rab32/38-KO B16-F1 cells. The graph shows the means and SEM of the data obtained in three independent experiments. ***, p < 0.001; NS, not significant (Tukey’s test). (E) Representative images of Tyr and Tyrp1 in WT cells and Rab32/38-KO B16-F1 cells, and their corresponding bright-field images. Scale bars, 20 μm. (F) Representative images of Tyr in melan-a cells and Rab32-deficient melan-cht cells, and their corresponding bright-field images. Scale bars, 20 μm.