Figure 4. Arl8b regulates RUFY1... | Rockefeller University Press

Figure 4.

$Arl8b regulates RUFY1 endosomal localization and promotes the interaction of RUFY1 and Rab14. (A and B) Representative confocal micrographs of HeLa cells treated with the indicated siRNA, followed by immunostaining for endogenous proteins (as labeled). (C) Representative confocal micrograph of HeLa cells treated with Arl8b siRNA and transfected with the untagged-Arl8b (Rescue) construct followed by immunostaining for RUFY1 and Arl8b. Single-channel images of RUFY1, EEA1, and Rab14 are shown as inverted images. Non-specific nuclear staining was observed with anti-Rab14 antibodies. Arrowheads (red for the RUFY1 channel and green for the EEA1/Rab14 channel) mark the colocalized pixels. Bars: (main) 10 µm; (insets) 2 µm. (D) Quantification of the number of RUFY1 punctae in HeLa cells upon different siRNA treatments as indicated. 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; n.s., not significant; unpaired two-tailed t test). (E) HeLa cells treated with either control or Arl8b siRNA were homogenized and subjected to ultracentrifugation to separate membrane and cytosol fractions. The supernatant is referred to as cytosol, and the pellet fraction was further treated with 1% Triton X (TX)-100 followed by ultracentrifugation to separate TX-100-insoluble membranes obtained as pellets and supernatant as TX-100-soluble fractions. Cytosol, TX-100 soluble and insoluble pellets were separated by SDS-PAGE followed by immunoblotting with indicated antibodies. Note: “*” marks the non-specific band observed at ∼71 kD upon immunoblotting with anti-RUFY1 antibody. The detection of a non-specific band at ∼71 kD by this antibody was further confirmed by RUFY1 siRNA as shown in Fig. S2 L. (F) Densitometric analysis of RUFY1 band signal in TX-100 insoluble pellet normalized to the input signal. The values plotted are the averages from two independent experiments. (G) Lysates of HeLa cells treated with indicated siRNA were immunoprecipitated with anti-Rab14 antibodies, and the precipitates were IB with the indicated antibodies. (H) Densitometric analysis of RUFY1 band intensity normalized to input and to direct IP of Rab14. The values plotted are the mean ± SD from three independent experiments (***P < 0.001; unpaired two-tailed t test). (I) Recombinant GST-RUFY1 (WT) protein was incubated with MBP alone or GDP/GTP-loaded MBP-Rab14, immobilized on amylose resin, in the presence of increasing amounts of His-Arl8b (WT) or His-Rab7 (WT). The precipitates were IB with the indicated antibodies, and Ponceau S staining was done to visualize the purified proteins. Source data are available for this figure: SourceData F4.$

Arl8b regulates RUFY1 endosomal localization and promotes the interaction of RUFY1 and Rab14. (A and B) Representative confocal micrographs of HeLa cells treated with the indicated siRNA, followed by immunostaining for endogenous proteins (as labeled). (C) Representative confocal micrograph of HeLa cells treated with Arl8b siRNA and transfected with the untagged-Arl8b (Rescue) construct followed by immunostaining for RUFY1 and Arl8b. Single-channel images of RUFY1, EEA1, and Rab14 are shown as inverted images. Non-specific nuclear staining was observed with anti-Rab14 antibodies. Arrowheads (red for the RUFY1 channel and green for the EEA1/Rab14 channel) mark the colocalized pixels. Bars: (main) 10 µm; (insets) 2 µm. (D) Quantification of the number of RUFY1 punctae in HeLa cells upon different siRNA treatments as indicated. 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; n.s., not significant; unpaired two-tailed t test). (E) HeLa cells treated with either control or Arl8b siRNA were homogenized and subjected to ultracentrifugation to separate membrane and cytosol fractions. The supernatant is referred to as cytosol, and the pellet fraction was further treated with 1% Triton X (TX)-100 followed by ultracentrifugation to separate TX-100-insoluble membranes obtained as pellets and supernatant as TX-100-soluble fractions. Cytosol, TX-100 soluble and insoluble pellets were separated by SDS-PAGE followed by immunoblotting with indicated antibodies. Note: “*” marks the non-specific band observed at ∼71 kD upon immunoblotting with anti-RUFY1 antibody. The detection of a non-specific band at ∼71 kD by this antibody was further confirmed by RUFY1 siRNA as shown in Fig. S2 L. (F) Densitometric analysis of RUFY1 band signal in TX-100 insoluble pellet normalized to the input signal. The values plotted are the averages from two independent experiments. (G) Lysates of HeLa cells treated with indicated siRNA were immunoprecipitated with anti-Rab14 antibodies, and the precipitates were IB with the indicated antibodies. (H) Densitometric analysis of RUFY1 band intensity normalized to input and to direct IP of Rab14. The values plotted are the mean ± SD from three independent experiments (***P < 0.001; unpaired two-tailed t test). (I) Recombinant GST-RUFY1 (WT) protein was incubated with MBP alone or GDP/GTP-loaded MBP-Rab14, immobilized on amylose resin, in the presence of increasing amounts of His-Arl8b (WT) or His-Rab7 (WT). The precipitates were IB with the indicated antibodies, and Ponceau S staining was done to visualize the purified proteins. Source data are available for this figure: SourceData F4.

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