Fig. 5
c-Kit-mediated phospho-PHBY259 in the membrane raft associates with Notch3 to regulate the Notch3—PBX1 signaling pathway. a Analysis of Notch3 (FL & NTM) and PBX1 protein expression in SKOV3_vector and SKOV3_c-Kit cells by western blotting. The quantification data represent the mean ± SD of three independent experiments. **, P < 0.01; *, P < 0.05, t test. b SKOV3 cells were transfected with empty vector, wild-type pD-PHB-HA, or mutant PHB plasmid (pD-PHBY259F-HA) for 48 h. The levels of Notch3 (FL & NTM) and PBX1 were analyzed by western blotting. c Transfection with empty vector or mutant PHB plasmid (pD-PHBY259F-HA) in SKOV3_c-Kit or SKOV3GL-G4 cells for 48 h. The levels of Notch3 (FL & NTM) and PBX1 were analyzed in whole cell lysate (WCL, top). Endo-PHBY259, PHB and exo-PHB expression was analyzed in the lipid raft domain (bottom) by western blotting. d SKOV3 cells transfected with mutant PHB plasmid (pD-PHBY259F-HA) were treated with or without chloroquine (25 μM) (or MG132; 20 μM), and the level of Notch3 was determined by western blotting and compared to that in SKOV3 cells transfected with wild-type pD-PHB-HA plasmid. e To monitor protein stability, SKOV3 cells transfected with wild-type pD-PHB-HA or mutant PHB plasmid (pD-PHBY259F-HA) were treated with cycloheximide (CHX; 50 μM), and the level of Notch3 was measured at the indicated time points. Data represent the mean ± SD of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05, t test. f SKOV3_c-Kit cells were transiently transfected with plasmid pD-PHB-HA, and immunoprecipitation was performed with anti-HA tag antibody. Coimmunoprecipitation (Co-IP) proteins were detected by western blotting with anti-c-Kit, anti-PHB, and anti-Notch3 antibodies. g Localization of c-Kit, raft-PHB, and Notch3 was analyzed in SKOV3_c-Kit cells through confocal microscopy. Scale bar = 10 μm. h SKOV3 cells were transfected with empty vector, wild-type pD-PHB-HA, or mutant PHB plasmid (pD-PHBY259F-HA) for 48 h. PHB was immunoprecipitated with anti-HA antibody, and coimmunoprecipitation of Notch3 was detected by western blotting. Blots are representative of three independent experiments