Fig. 5

NEURL3 promotes K48-linked ubiquitination and degradation of Vimentin. (a) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with Myc-NEURL3 or its empty vector, together with Flag-Vimentin plasmid and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K63O-Ub). (b) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with shNEURL3 plasmid or its control vector, together with Flag-Vimentin plasmid and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K48R-Ub). (c) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with empty vector, wild-type (WT) Myc-NEURL3 or its C217Y mutant, together with Flag-Vimentin and HA-WT-Ub. (d-e) Representative images and greyscale analyses of Vimentin protein levels after CHX treatment in SUNE1 (d) and 293T (e) cells co-transfected with WT Myc-NEURL3 or its C217Y mutant, together with Flag-Vimentin. (f) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with Myc-NEURL3 or its empty vector, together with WT Flag-Vimentin or its K97R mutant and HA-WT-Ub. (g-h) Representative images and greyscale analyses of Vimentin protein levels after CHX treatment in SUNE1 (g) and 293T (h) cells co-transfected with Myc-NEURL3, together with WT Flag-Vimentin or its K97R mutant. Data of d, e, g, and h are shown as mean ± SD, and the p-values were determined by Student’s t-test (ns, no significance; *p < 0.05)