Perinuclear positioning of RUFY1, CI-M6PR, and Rab14 is dependent on dynein. (A) HEK293T cell lysates were immunoprecipitated with either anti-RSK1 (used as a control) or anti-RUFY1 antibodies bound to Protein A/G beads. The precipitates were immunoblotted (IB) with the indicated antibodies. (B) AlphaFold prediction of structures for human proteins-RUFY1, HOOK3 and BICD2. (C, D, G, and H) Representative immunofluorescence images of HeLa cells treated with either control or Dynein Heavy Chain (DHC) siRNA, followed by immunostaining with anti-RUFY1 and anti-CI-M6PR antibodies (C and D) or anti-Rab14 and anti-CI-M6PR antibodies (G and H). The inset indicates colocalized pixels. Bars: (main) 10 µm; (insets) 2 µm. (E, F, and I) The graph shows a radial profile plot of RUFY1 (E), CI-M6PR (F), and Rab14 (I) intensity in HeLa cells treated with either control or DHC siRNA. The values plotted are the mean ± SD from three independent experiments with 25 cells (E and F) and 15–20 cells (I) analyzed per experiment. (J–M) Representative confocal images of HeLa cells co-transfected with plasmids expressing 2x-FKBP-GFP-RUFY1 (WT or mutants as labeled) and DsRed-Rab14. Bars: (main) 10 µm; (insets) 2 µm. Source data are available for this figure: SourceData FS5.