Figure S3.
Rab14, but not Arl8b, determines RUFY1 recruitment to membranes. (A–D) Confocal micrographs of HeLa cells transfected with C-terminal Mito-HA-tagged GTP-locked versions of small G proteins, Arl8b (Q75L; A), Rab14 (Q70L; B), Rab4a (Q72L; C), and Rab5 (Q79L; D). Cells were fixed and immunostained with anti-HA and anti-Tom20 (mitochondrial marker) antibodies. Bars: 10 µm. (E–I) Confocal micrographs of HeLa cells co-transfected with Mito-tagged GTP-locked versions of small G proteins as described above and with either FLAG-SKIP (E) or RUFY1-FLAG (F–I). Cells were fixed and immunostained with anti-HA and anti-FLAG antibodies. Bars: 10 µm. (J) Pearson’s colocalization coefficient quantification of RUFY1-FLAG and Mito-HA-tagged GTP-locked versions of small G proteins-Rab4a, Rab5, Rab14, and Arl8b. 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. (K) Control- and Rab14-siRNA-treated HeLa cell lysates were immunoblotted (IB) with anti-Rab14 antibody for assessing the knockdown efficiency. α-tubulin was used as the loading control. (L) Representative confocal micrographs of HeLa cells treated with either control or Rab14 siRNA followed by immunostaining for endogenous RUFY1. Bars: 10 µm. (M) Pearson’s colocalization coefficient (PCC) was quantified for RUFY1-GFP and DsRed-tagged-Rab14 signals in either control or Arl8b siRNA-treated HeLa cells. PCC was calculated from a single frame of live-cell imaging videos. The values plotted are the mean ± SD from two independent experiments with 16–17 cells analyzed per treatment (****P < 0.0001; unpaired two-tailed t test). (N) Yeast two-hybrid assay. The indicated yeast co-transformants were spotted (five-fold serial dilution) on -Leu/-Trp (non-selection) and -Leu/-Trp/-His/-Ade (selection) media plates to confirm viability and interactions, respectively. Source data are available for this figure: SourceData FS3.

Rab14, but not Arl8b, determines RUFY1 recruitment to membranes. (A–D) Confocal micrographs of HeLa cells transfected with C-terminal Mito-HA-tagged GTP-locked versions of small G proteins, Arl8b (Q75L; A), Rab14 (Q70L; B), Rab4a (Q72L; C), and Rab5 (Q79L; D). Cells were fixed and immunostained with anti-HA and anti-Tom20 (mitochondrial marker) antibodies. Bars: 10 µm. (E–I) Confocal micrographs of HeLa cells co-transfected with Mito-tagged GTP-locked versions of small G proteins as described above and with either FLAG-SKIP (E) or RUFY1-FLAG (F–I). Cells were fixed and immunostained with anti-HA and anti-FLAG antibodies. Bars: 10 µm. (J) Pearson’s colocalization coefficient quantification of RUFY1-FLAG and Mito-HA-tagged GTP-locked versions of small G proteins-Rab4a, Rab5, Rab14, and Arl8b. 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. (K) Control- and Rab14-siRNA-treated HeLa cell lysates were immunoblotted (IB) with anti-Rab14 antibody for assessing the knockdown efficiency. α-tubulin was used as the loading control. (L) Representative confocal micrographs of HeLa cells treated with either control or Rab14 siRNA followed by immunostaining for endogenous RUFY1. Bars: 10 µm. (M) Pearson’s colocalization coefficient (PCC) was quantified for RUFY1-GFP and DsRed-tagged-Rab14 signals in either control or Arl8b siRNA-treated HeLa cells. PCC was calculated from a single frame of live-cell imaging videos. The values plotted are the mean ± SD from two independent experiments with 16–17 cells analyzed per treatment (****P < 0.0001; unpaired two-tailed t test). (N) Yeast two-hybrid assay. The indicated yeast co-transformants were spotted (five-fold serial dilution) on -Leu/-Trp (non-selection) and -Leu/-Trp/-His/-Ade (selection) media plates to confirm viability and interactions, respectively. Source data are available for this figure: SourceData FS3.

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