Figure 8.
RUFY1 interacts with the dynein-dynactin complex and acts as a dynein cargo adaptor. (A) HEK293T cell lysates were immunoprecipitated using anti-RUFY1 antibody bound to Protein-A/G beads, and the precipitates were immunoblotted (IB) with indicated antibodies. (B) Recombinant GST and GST-LIC1WT (389–523 a.a.) proteins were immobilized on glutathione-coated-agarose beads and incubated with HEK293T cell lysates expressing RUFY1 (WT)-FLAG or RUFY1 (ΔRUN)-FLAG. The precipitates were IB with anti-FLAG antibody and Ponceau S staining was done to visualize the purified proteins. (C) Recombinant GST, GST-LIC1WT, and GST-LIC1FFAA (F447A/F448A) proteins were immobilized on glutathione-coated-agarose beads and incubated with HEK293T cell lysates expressing RUFY1 (WT)-FLAG. The precipitates were IB with anti-FLAG antibody and Ponceau S staining was done to visualize the purified proteins. (D) Densitometric analysis of RUFY1 (WT) pulldown using the indicated GST-fusion proteins (normalized to input signals) is shown. The values plotted are the mean ± SD from three independent experiments (***P < 0.001; unpaired two-tailed t test). (E–H) Representative confocal images of HeLa cells transiently expressing FRB-Tom70p with 2x-FKBP-GFP (E and F) or 2x-FKBP-GFP-RUFY1 (G and H) treated with or without rapamycin, followed by immunostaining with anti-Tom20 antibodies to visualize mitochondria. A white boundary marks the co-transfected cells. Bars: 10 µm. (I) The Tom20 signal intensity profile was quantified with respect to distance from the nucleus of HeLa cells expressing indicated FRB-FKBP fusion proteins upon addition of rapamycin. 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 or bottom of each data set (****P < 0.0001; unpaired two-tailed t test). (J and K) Representative confocal micrographs of HeLa cells treated with the indicated siRNAs and co-transfected with FRB-Tom70p and 2x-FKBP-GFP-RUFY1 constructs, followed by 2 h treatment with rapamycin before fixation. Cells were immunostained with an anti-Tom20 antibody to visualize mitochondria. A white boundary marks the co-transfected cells. Bars: 10 µm. (L) The Tom20 signal intensity profile was quantified with respect to distance from the nucleus of HeLa cells treated with indicated siRNA and expressing FRB-FKBP fusion proteins in the presence of rapamycin. 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 or bottom of each data set (****P < 0.0001; unpaired two-tailed t test). Source data are available for this figure: SourceData F8.

RUFY1 interacts with the dynein-dynactin complex and acts as a dynein cargo adaptor. (A) HEK293T cell lysates were immunoprecipitated using anti-RUFY1 antibody bound to Protein-A/G beads, and the precipitates were immunoblotted (IB) with indicated antibodies. (B) Recombinant GST and GST-LIC1WT (389–523 a.a.) proteins were immobilized on glutathione-coated-agarose beads and incubated with HEK293T cell lysates expressing RUFY1 (WT)-FLAG or RUFY1 (ΔRUN)-FLAG. The precipitates were IB with anti-FLAG antibody and Ponceau S staining was done to visualize the purified proteins. (C) Recombinant GST, GST-LIC1WT, and GST-LIC1FFAA (F447A/F448A) proteins were immobilized on glutathione-coated-agarose beads and incubated with HEK293T cell lysates expressing RUFY1 (WT)-FLAG. The precipitates were IB with anti-FLAG antibody and Ponceau S staining was done to visualize the purified proteins. (D) Densitometric analysis of RUFY1 (WT) pulldown using the indicated GST-fusion proteins (normalized to input signals) is shown. The values plotted are the mean ± SD from three independent experiments (***P < 0.001; unpaired two-tailed t test). (E–H) Representative confocal images of HeLa cells transiently expressing FRB-Tom70p with 2x-FKBP-GFP (E and F) or 2x-FKBP-GFP-RUFY1 (G and H) treated with or without rapamycin, followed by immunostaining with anti-Tom20 antibodies to visualize mitochondria. A white boundary marks the co-transfected cells. Bars: 10 µm. (I) The Tom20 signal intensity profile was quantified with respect to distance from the nucleus of HeLa cells expressing indicated FRB-FKBP fusion proteins upon addition of rapamycin. 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 or bottom of each data set (****P < 0.0001; unpaired two-tailed t test). (J and K) Representative confocal micrographs of HeLa cells treated with the indicated siRNAs and co-transfected with FRB-Tom70p and 2x-FKBP-GFP-RUFY1 constructs, followed by 2 h treatment with rapamycin before fixation. Cells were immunostained with an anti-Tom20 antibody to visualize mitochondria. A white boundary marks the co-transfected cells. Bars: 10 µm. (L) The Tom20 signal intensity profile was quantified with respect to distance from the nucleus of HeLa cells treated with indicated siRNA and expressing FRB-FKBP fusion proteins in the presence of rapamycin. 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 or bottom of each data set (****P < 0.0001; unpaired two-tailed t test). Source data are available for this figure: SourceData F8.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal