Fig. 4

NEURL3 interacts with Vimentin to promote its degradation. (a) Silver staining of SDS-PAGE gels showing HA immunoprecipitates that were pulled down from SUNE1 cells transfected with HA-NEURL3 plasmid. The interest proteins are indicated. (b) Co-IP with anti-HA or Vimentin antibodies showing the interactions between HA-NEURL3 and endogenous Vimentin in HONE1 and SUNE1 cells. (c) IF staining showing co-localization of exogenous HA-NEURL3 and endogenous Vimentin in HONE1 and SUNE1 cells (Scale, 50 μm). (d) Schematic diagram showing the structure of Vimentin or its deletion mutant plasmids. (e) Co-IP revealing interactions of NEURL3 with different Vimentin mutants in 293T cells. (f-g) Vimentin protein levels in HONE1 and SUNE1 cells transfected with gradient concentrations of HA-tagged NEURL3 plasmid (f), as well as shNEURL3 plasmids or its control vector (g). (h) IF staining showing Vimentin protein levels in HONE1 and HK1 cells transfected with HA-NEURL3 plasmid or its vector control (Scale bar, 50 µM). (i-j) Representative images and greyscale analyses of Vimentin protein levels in SUNE1 and 293T cells transfected with HA-NEURL3 plasmid or its vector control (i), as well as shNEURL3 plasmid or its control vector (j), after the CHX treatment. (k-l) Vimentin protein levels in HONE1 and SUNE1 cells transfected with HA-NEURL3 plasmid or its vector control after treated with MG132 (k) and CQ (l). Data of i and j are shown as mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)