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. 2020 Jun 17;10:9868. doi: 10.1038/s41598-020-66789-x

Figure 5.

Figure 5

Rps3 is required for HECTD1 to ubiquitinate IκBα. (A) HIEC cells were transfected with siRps3 for 72 h, and then the cell lysates were obtained for immunoprecipitation with anti-LXN and anti-HECTD1 antibody, respectively. The immunoblotting was performed with antibody as indicated. (B) Protein samples from LXN+/+ (WT) and LXN−/− (KO) mice colon tissue were immunoprecipitated with anti-HECTD1 and anti-IgG antibody, and then the complex was separated by 10% SDS-PAGE, and stained with silver staining (left panel). Transferred membrane was immunoblotted with antibodies as indicated (right panel). (C) Cell lysates from HCT116 cells were immunoprecipitated with anti-HECTD1 antibody or control IgG, and then the purified protein complex was separated by 10% SDS-PAGE. Transferred membrane was immunoblotted with anti-ubiquitin (left panel), anti-HECTD1 and anti-IkBα antibodies, as indicated (right panel). (D) HEK293T cells were co-transfected with His-IκBα, HA-Ub and HA-HECTD1 plasmids as indicated. For ubiquitination assay, the cells were incubated in the presence of 10 μM MG132 for 12 h before assay. 48 h after co-transfection, immunoprecipitation was performed with anti-His antibody, and ubiquitylation was detected by Western blot. (E) HCT116 cells were co-transfected with His-IκBα, HA-Ubiquitin plasmid plus HECTD1 siRNA for 72 h. Cell lysates were subjected to immunoprecipitation with anti-His antibody, and then the ubiquitylation of His-IκBα was determined by western blot. (F) 72 h after transfection of either HECTD1 siRNA or Rps3 siRNA, HCT116 cells were stimulated with TNF-α (20 ng/mL) for 30 min, cell lysates were subjected to immunoprecipitation with anti-IκBα antibody followed by immunoblot analysis with anti-Ub and anti-IκBα. (G) HEK293T cells were co-transfected with His-IκBα, HA-HECTD1, wild-type HA-Ub (WT) or Ub mutant plasmids as indicated. 48 h after transfection, cell lysates were immunoprecipitated with anti-His antibody and subjected to immunoblotting with anti-HA antibody. (H) HEK293T cells were co-transfected with His-IκBα, HA-HECTD1, wild-type HA-Ub (WT) or Ub mutant plasmids (K48R). 48 h after transfection, cell lysates were immunoprecipitated with anti-His antibody and subjected to immunoblotting with K48-linkage specific ubiquitin antibody. Data are representative of three independent experiments.