Fig. 5

HURP p725 regulates the localization and protein stability of TRIP11. A Interaction of GFP-TRIP11 with HA-HURP. 293T cells transfected with GFP-TRIP11 and various versions of HA-HURP were analyzed for the interaction of TRIP11 and HURP by performing immune-coprecipitation. pcDNA is the empty vector pcDNA3.1. Three independent experiments were performed. B Interaction of HURP p725 and TRIP11. Immuno-coprecipitation was conducted in HeLa cells to examine the interaction of endogenous TRIP11 and HURP p725 or np725. Three independent experiments were performed. C HURP was required for maintaining the GA localization of TRIP11 and not the vice versa. HeLa cells with shHURP or TRIP11 shRNA (shTRIP11) were analyzed for the knockdown efficiency (lower) or subcellular distribution of HURP p725 or TRIP11 (upper). Three independent experiments were performed. D Translocation of TRIP11 to nucleus did not affect subcellular localization of HURP p725. HeLa cells treated with solvent or T3 (2 μM, 2 h) were surveyed for the subcellular distribution of HURP p725 or TRIP11. Three independent experiments were performed. E HURP employed its N-terminal to interact with TRIP11. 293 cells, transfected with GFP-TRIP11 and each one of empty vector pCDNA3.1, HA tagged HURP full length (FL), 1–300 or 300–846, were analyzed for the interaction with TRIP11 by conducting immune-coprecipitation using antibodies against HA. Three independent experiments were performed. F The nuclear localization of HURP 1–300 attracted TRIP11 into nucleus. HeLa cells, harboring HA-HURP FL, 1–300, 300–846, were examined for subcellular localization of TRIP11 by performing immunofluorescence. Three independent experiments were performed. G The HURP deletion mutant 1–300 attracting TRIP11 into the nucleus disassembled GA. HeLa cells harboring HURP full length (FL), 1–300 or 300–846 were examined for GA structure by conducting immunofluorescence adopting antibodies against GRASP55. To analyze the structure of GA stained by GRAST65 antibodies, 200 cells either with pcDNS3.1, HA-HURP 1–300, 300–846 or FL, for each independent experiment were examined, and 3 independent experiments were performed. H Knockdown of HURP destabilized TRIP11. HeLa cells with shLuc or shHURP were treated with cycloheximide (CHX) to inhibit protein de novo synthesis for 0–9 h. Western blots were then followed to determine the protein stability of TRIP11. Three independent experiments were conducted. *, **, and *** stand for statistical significance by Student’s t-test with p < 0.05, 0.01 and 0.001 respectively. Scale bar: 10 μm