Fig. 5: RUFY3 links Arl8b to the JIP4-dynein complex.

a, b GST-pulldown assay of semi-purified FLAG-tagged-JIP4 with GST and GST-RUFY3 and immunoblotted (IB) with anti-FLAG antibody. GST proteins were visualized by Ponceau S staining. Quantification of blots from two independent experiments is shown in b. c–f Lysates of HEK293T cells were subjected to endogenous IP as labeled and the precipitates were IB with the indicated antibodies. Quantification of the blots is shown in d, f. The values plotted are the mean ± SD from three independent experiments. For d, **p = 0.0052 and *p = 0.0236 and for f, **p = 0.0015 (p150); **p = 0.0043 (DIC), and *p = 0.0251 (two-tailed Student’s t-test). g–i Confocal micrographs of HeLa cells transfected with Arl8b-HA (g) or co-transfected with RUFY3 (UT) (h) and stained with indicated antibodies. Transfected cells are outlined, and some panels are shown in an inverted grayscale. The colocalization of JIP4 with Arl8b was measured by Pearson’s correlation coefficient (i). The values plotted are the mean ± SD from three independent experiments. Experiments are color-coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the graph (****p < 0.0001; two-tailed Student’s t-test). j, k HEK293T cell lysates expressing Arl8b-HA or co-expressing Arl8b-HA and RUFY3-FLAG were subjected to IP and the precipitates were IB with the indicated antibodies. Quantification of the blot is shown in k and values plotted are mean ± SD from three independent experiments (**p = 0.0045; two-tailed Student’s t-test). l, m HEK293T cells were treated with the indicated siRNAs and subjected to endogenous IP using an anti-Arl8 antibody. The precipitates were IB with the indicated antibodies. The quantification of the blot is shown in m and values plotted are the mean ± SD from three independent experiments (****p < 0.0001; two-tailed Student’s t-test). n, o Confocal images of HeLa cells treated with indicated siRNAs and transfected with RUFY3-HA. The cells were stained with anti-LAMP1 and anti-HA antibodies, respectively. Transfected cells are outlined, and some panels are shown in an inverted grayscale. p The distribution of lysosomes was quantified from the experiments shown in n, o. The values plotted are the mean ± SD from three independent experiments. The total number of cells analyzed is indicated on the graph (****p < 0.0001; two-tailed Student’s t-test). Scale Bars: 10 µm.