Fig. 6

ENO1 interacts with YWHAZ and promotes its protein stability. A Total cell lysate was extracted from H929 cells, purified and resolved on SDS-PAGE. Coomassie brilliant blue stained gel exhibited differential bands, and the bands were retrieved and analysed by mass spectrometry. B Venn diagram showed that 195 proteins specifically pulled down from the IP group, excluding the 97 proteins that co-crossed between the IgG and IP groups. C Identified YWHAZ peptides are shown. D Endogenous YWHAZ in MM cells was immunoprecipitated using anti-ENO1 antibody with rabbit IgG as a nonspecific control. E HEK293T cells were transfected with Flag-YWHAZ, HA-ENO1, or both constructs. Cell lysates were immunoprecipitated using anti-Flag and the immunoprecipitants or input were analysed by immunoblotting with anti-HA or anti-Flag. F Co-localization between YWHAZ (green) and ENO1 (red) was analysed by confocal microscopy in H929 cells. (Scale bar: 5 μm). G Co-immunoprecipitation was conducted to investigate the interaction between YWHAZ and HA-vector, full‐length HA‐ENO1, and truncated forms of HA‐ENO1 in HEK293T cells. H In the case of ENO1 knockdown, YWHAZ and ENO1 protein levels were analysed by Western blot. Actin was used as the loading controls. I Downregulation of ENO1 increases YWHAZ degradation. Western blot detected the alteration of YWHAZ in H929 cells with treatment of 10 µg/mL CHX for the indicated times. Actin was used as a loading control