Fig. 6

HURP p725 binds and stabilizes more cis-GAFs. A HURP p725 interacted with three cis-Golgi proteins. HeLa cells were applied to HURP p725 antibodies or np725 antibodies-based immuno-coprecipitation, and Western blots adopting antibodies against GRASP65, GM130, or GP73 were performed. Three independent experiments were performed. B HURP p725 did not bind GRASP55. Immuno-coprecipitation was performed in HeLa cells harboring empty vector or Myc-GRASP55 to detect the interaction of GRASP55 and HURP p725 or np725. Three independent experiments were performed. C Interaction of EGFP-HURP with the three cis-GAFs. 293 cells harboring EGFP, EGFP-HURP WT, 725E or 725A were analyzed for the interaction of EGFP-HURP with GRASP65 (I), GM130 (II) and GP73 (III). Three independent experiments were performed. D Knockdown of HURP destabilized GRASP65, GM130 and GP73, while did not affect GRASP55. Protein stability of the four Golgi proteins was surveyed in shLuc and shHURP cells treated with cycloheximide. Three independent experiments were conducted to obtain the relative protein level of each protein. *, **, and *** stand for statistical significance by Student’s t-test with p < 0.05, 0.01 and 0.001 respectively