Fig. 4: FBXO28 interacts with, and ubiquitylates, SNAI2.

A, B Huh7 cells were transfected with indicated plasmids for 72 h, and then subjected to IP and IB analyses. C Huh7 cells were subjected to IP with FBXO28 antibody or normal rabbit IgG and IB analyses. D Co-localization of FBXO28 and SNAI2 by immunofluorescence staining in Hep3b cells. Representative images are shown. Scale bar: 8 μm. E HEK293T cells co-expressing FBXO28 and full-length or deletion mutants of SNAI2 (with schematic diagram shown on the upper panel in F) were cultured for 48 h prior to IP and IB analyses. F Upper panel: A schematic representation of SNAI2 deletion mutants. Lower panel: HEK293T cells transfected with indicated plasmids for 48 h were subjected to IB analyses. Arrows indicate the target band. G In vitro ubiquitination assay using cell immunocomplex pulled down with anti-Myc beads from HEK293T cell extracts after transfection with FBXO28-Flag plasmid for 48 h. The polyubiquitination was detected by IB with anti-SNAI2 antibody. H HEK293T cells were co-transfected with indicated plasmids for 72 h. The polyubiquitylated proteins were purified by Ni-NTA beads and detected with anti-HA antibody. I HEK293T cells were transfected with indicated plasmids for 72 h followed by Ni-NTA beads purification and IB with anti-HA antibody. J HEK293T cells were transfected with indicated plasmids for 48 h prior to IP and IB analyses. SE short exposure. LE long exposure.