Fig. 7

ARF1/Golgin-160 controls subcellular distribution of HURP p725. A BFA disorganized HURP p725 and GA. HeLa cells treated with or without 5 μM BFA for 1 h were visualized for GA morphology (BRAST65) or subcellular distribution (I) and protein level (II) of HURP p725 by immunofluorescence and Western blot. Three independent experiments were performed. B Native gel analysis of HURP and HURP p725. HeLa cells transfected EGFP, EGFP-HURP (I), or pcDNA3.1 empty vector, HA-HURP (II, III) were treated with or without BFA. The resulting samples were applied to native gel-based electrophoresis followed by performing Western blots to detect GFP (I), HURP (II) or HURP p725 (III). Three independent experiments were performed. C Gel filtration assay for HURP. HeLa cells transfected with EGFP or EGFP-HURP were treated with or without BFA. Subsequently, the cell extracts of fraction 5–18 from gel filtration were collected and Western blots adopting GFP antibodies were performed. Three independent experiments were performed. D Knockdown of ARF1 or GBF1 disorganized HURP p725. HeLa cells harboring shLuc, ARF1 shRNA (shARF1) or GBF1 shRNA (shGBF1) were examined for the knockdown efficiency of the two proteins by Western blots (I) or the subcellular distribution of HURP p725 by immunofluorescence (II). The % of cells with each type of HURP p725 distribution was counted and plotted (III). To analyze the distribution of HURP p725, 200 cells either with shLuc, shARF1 or GBF1 for each independent experiment were examined, and 3 independent experiments were performed. E ARF1 did not interact with HURP p725. Immuno-coprecipitation was performed to detect the interaction of HA-tagged ARF1 WT or inactive mutant T31N with HURP p725. Three independent experiments were performed. F Dynein inhibitor disorganized HURP p725. HeLa cells treated with 50 μM dynein inhibitor Cilliobrevin D for 0–16 h were applied to immunofluorescence to detect subcellular localization of HURP p725. The % of cells with each kind of HURP p725 distribution was counted and plotted. To analyze the distribution pattern of HURP p725, 200 cells treated with Cilliobrevin for different times for each independent experiment were examined, and 3 independent experiments were performed. G Knockdown of Golgin-160 dispersed HURP p725. HeLa cells harboring shLuc or Golgi-160 shRNA (shGolgin-160) were examined for HURP p725 distribution (I) or knockdown down efficiency (II). The % of cells with each kind of HURP p725 distribution was counted and plotted (III). To analyze the distribution pattern of HURP p725, 200 cells either with shLuc or shGolgin-160 for each independent experiment were examined, and 3 independent experiments were performed. H Golgin-160 interacted with HURP p725. Immuno-coprecipitation employing antibodies against HURP p725 or np725 was conducted in HeLa cells to examine the interaction of HURP and Golgin-160. Three independent experiments were performed. *, **, and *** stand for statistical significance by Student’s t-test with p < 0.05, 0.01 and 0.001 respectively. Scale bar: 10 μm