Figure 1.
RUFY1 isoforms interact with the GTP-bound form of Arl8b. (A) Schematic representation of the domain architecture of RUFY1 isoforms. (B) Lysates of HeLa and HEK293T cells treated with indicated siRNA (control siRNA, RUFY1 siRNA #1, #2, and SP [SMARTpool]) were immunoblotted (IB) with anti-RUFY1 antibody for assessing the specificity of the antibody and with anti-α-tubulin antibody as a loading control. Arrowheads indicate the two isoforms (Rabip4 and Rabip4′). (C) Recombinant GST and GST-Arl8b proteins were immobilized on glutathione-coated-agarose beads and loaded with either GTP or GDP and then incubated with HEK293T cell lysates expressing Rabip4′-FLAG (longer isoform) or Rabip4-FLAG (shorter isoform). The precipitates were IB with anti-FLAG antibody, and Ponceau S staining was done to visualize the purified proteins. (D) RUFY1-FLAG (longer isoform) was co-transfected with vector or with different forms of Arl8b-HA into HEK293T cells, and the lysates were immunoprecipitated with anti-HA antibodies-conjugated-agarose beads. The precipitates were IB with the indicated antibodies. (E) HeLa cell lysates were immunoprecipitated with anti-Arl8a/b antibodies-conjugated-agarose beads. The precipitates were IB with the indicated antibodies. (F) Representative confocal micrograph of HeLa cells immunostained for both endogenous RUFY1 and Arl8b. (G–I) Representative confocal micrographs of HeLa cells transfected with Arl8b (WT)-HA, Arl8b (Q75L)-HA, and Arl8b (T34N)-HA, followed by immunostaining with anti-HA and anti-RUFY1 antibodies. For F–I, arrowheads in the insets mark the colocalized pixels. Bars: (main) 10 µm; (insets) 2 µm. (J) Pearson’s and Mander’s colocalization coefficient quantification of endogenous RUFY1 with endogenous Arl8b. (K) Pearson’s correlation coefficient quantification of endogenous RUFY1 with different forms of transfected Arl8b-HA. For graphs (J and K), 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). Source data are available for this figure: SourceData F1.

RUFY1 isoforms interact with the GTP-bound form of Arl8b. (A) Schematic representation of the domain architecture of RUFY1 isoforms. (B) Lysates of HeLa and HEK293T cells treated with indicated siRNA (control siRNA, RUFY1 siRNA #1, #2, and SP [SMARTpool]) were immunoblotted (IB) with anti-RUFY1 antibody for assessing the specificity of the antibody and with anti-α-tubulin antibody as a loading control. Arrowheads indicate the two isoforms (Rabip4 and Rabip4′). (C) Recombinant GST and GST-Arl8b proteins were immobilized on glutathione-coated-agarose beads and loaded with either GTP or GDP and then incubated with HEK293T cell lysates expressing Rabip4′-FLAG (longer isoform) or Rabip4-FLAG (shorter isoform). The precipitates were IB with anti-FLAG antibody, and Ponceau S staining was done to visualize the purified proteins. (D) RUFY1-FLAG (longer isoform) was co-transfected with vector or with different forms of Arl8b-HA into HEK293T cells, and the lysates were immunoprecipitated with anti-HA antibodies-conjugated-agarose beads. The precipitates were IB with the indicated antibodies. (E) HeLa cell lysates were immunoprecipitated with anti-Arl8a/b antibodies-conjugated-agarose beads. The precipitates were IB with the indicated antibodies. (F) Representative confocal micrograph of HeLa cells immunostained for both endogenous RUFY1 and Arl8b. (G–I) Representative confocal micrographs of HeLa cells transfected with Arl8b (WT)-HA, Arl8b (Q75L)-HA, and Arl8b (T34N)-HA, followed by immunostaining with anti-HA and anti-RUFY1 antibodies. For F–I, arrowheads in the insets mark the colocalized pixels. Bars: (main) 10 µm; (insets) 2 µm. (J) Pearson’s and Mander’s colocalization coefficient quantification of endogenous RUFY1 with endogenous Arl8b. (K) Pearson’s correlation coefficient quantification of endogenous RUFY1 with different forms of transfected Arl8b-HA. For graphs (J and K), 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). Source data are available for this figure: SourceData F1.

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