Figure S2.
RUFY1 and Arl8b colocalize on early/recycling endosomes and Arl8b regulates RUFY1 membrane localization. (A) Representative confocal micrograph of HeLa cells co-transfected with Arl8b-HA and RUFY1-GFP and incubated with Lysotracker Red (100 nM) for 2 h, followed by PFA fixation and immunostaining with anti-HA antibody. Bars: (main) 10 µm; (insets) 2 µm. (B–E) Structured illumination microscopy (SIM) of HeLa cells co-transfected with Arl8b-HA and RUFY1-GFP and immunostained for Arl8b using anti-HA antibodies and other endocytic markers as indicated. For B–E, arrowheads in the insets mark the colocalized pixels. The white arrowheads in the inset of Fig. S2 B mark Arl8b and Rab7 colocalized punctae, while the yellow arrowhead marks Arl8b and RUFY1 colocalized puncta. Bars: (main) 2 µm; (insets) 2 µm. (F) Control- and Arl8b-siRNA-treated HeLa cell lysates or lysates of WT- and Arl8b−/− knockout (KO)-HeLa cells were immunoblotted (IB) with anti-Arl8b antibody for assessing the knockdown efficiency. α-tubulin was used as the loading control. (G and H) Confocal images of wild-type (WT) and Arl8b−/− KO HeLa cells immunostained for endogenous RUFY1 and EEA1. Single-channel images of RUFY1 and EEA1 are represented as inverted images to facilitate understanding. Bars: 10 µm. (I) The graph shows the quantification of the number of RUFY1 punctae in WT and Arl8b−/− KO HeLa cells. 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 top of each data set (****P < 0.0001; unpaired two-tailed t test). (J) Representative confocal micrographs of HeLa cells transfected with Arl8b-HA followed by immunostaining with anti-HA and anti-RUFY1 antibodies. An asterisk denotes the Arl8b-HA-expressing cells. Bars: 10 µm. (K) The graph shows the quantification of RUFY1 punctae size in Arl8b-HA transfected and surrounding untransfected cells. 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 top of each data set (****P < 0.0001; unpaired two-tailed t test). (L) HeLa cell lysates from the indicated siRNA treatments were IB with anti-RUFY1 antibody (Santa Cruz Biotechnology [SCBT]; sc-398740) to assess the specificity of the antibody, and α-tubulin was used as the loading control. This antibody from SCBT recognizes the longer isoform of RUFY1 (Rabip4′; ∼80 kD; marked by an arrow) but also shows a non-specific band at ∼70 kD (marked by an asterisk) whose intensity is not reduced upon RUFY1 knockdown. Source data are available for this figure: SourceData FS2.

RUFY1 and Arl8b colocalize on early/recycling endosomes and Arl8b regulates RUFY1 membrane localization. (A) Representative confocal micrograph of HeLa cells co-transfected with Arl8b-HA and RUFY1-GFP and incubated with Lysotracker Red (100 nM) for 2 h, followed by PFA fixation and immunostaining with anti-HA antibody. Bars: (main) 10 µm; (insets) 2 µm. (B–E) Structured illumination microscopy (SIM) of HeLa cells co-transfected with Arl8b-HA and RUFY1-GFP and immunostained for Arl8b using anti-HA antibodies and other endocytic markers as indicated. For B–E, arrowheads in the insets mark the colocalized pixels. The white arrowheads in the inset of Fig. S2 B mark Arl8b and Rab7 colocalized punctae, while the yellow arrowhead marks Arl8b and RUFY1 colocalized puncta. Bars: (main) 2 µm; (insets) 2 µm. (F) Control- and Arl8b-siRNA-treated HeLa cell lysates or lysates of WT- and Arl8b−/− knockout (KO)-HeLa cells were immunoblotted (IB) with anti-Arl8b antibody for assessing the knockdown efficiency. α-tubulin was used as the loading control. (G and H) Confocal images of wild-type (WT) and Arl8b−/− KO HeLa cells immunostained for endogenous RUFY1 and EEA1. Single-channel images of RUFY1 and EEA1 are represented as inverted images to facilitate understanding. Bars: 10 µm. (I) The graph shows the quantification of the number of RUFY1 punctae in WT and Arl8b−/− KO HeLa cells. 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 top of each data set (****P < 0.0001; unpaired two-tailed t test). (J) Representative confocal micrographs of HeLa cells transfected with Arl8b-HA followed by immunostaining with anti-HA and anti-RUFY1 antibodies. An asterisk denotes the Arl8b-HA-expressing cells. Bars: 10 µm. (K) The graph shows the quantification of RUFY1 punctae size in Arl8b-HA transfected and surrounding untransfected cells. 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 top of each data set (****P < 0.0001; unpaired two-tailed t test). (L) HeLa cell lysates from the indicated siRNA treatments were IB with anti-RUFY1 antibody (Santa Cruz Biotechnology [SCBT]; sc-398740) to assess the specificity of the antibody, and α-tubulin was used as the loading control. This antibody from SCBT recognizes the longer isoform of RUFY1 (Rabip4′; ∼80 kD; marked by an arrow) but also shows a non-specific band at ∼70 kD (marked by an asterisk) whose intensity is not reduced upon RUFY1 knockdown. Source data are available for this figure: SourceData FS2.

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