Fig. 6: β-TrCP directly targets FOXN3 for ubiquitination and degradation.

a Sequence alignment across various species reveals a conserved DSGYAS motif located within the C-terminal domain of FOXN3. b A Co-IP assay was conducted in HEK293T cells to assess the interaction between Myc-tagged β-TrCP and Flag-tagged WT FOXN3 or FOXN3 lacking the DSGYAS motif (Δ411 − 416). The cells transfected with the indicated combinations were collected 48 hrs post-transfection. c Immunoblot analysis of Myc-tagged β-TrCP immunoprecipitated by Flag-tagged WT FOXN3 or its S412,416 A mutant form in HEK293T cells. The cells transfected with the indicated combinations were collected 48 hrs post-transfection. d A ubiquitination assay was conducted in HEK293T cells to examine the effect of β-TrCP overexpression on the ubiquitination of WT FOXN3 or its S412,416 A mutant. The cells transfected with Flag-tagged FOXN3, Myc-tagged β-TrCP, and HA-tagged ubiquitin were collected 48 h post-transfection. e A ubiquitination assay was conducted in HEK293T cells to examine the effect of β-TrCP knockdown on the ubiquitination of FOXN3. The cells transfected with Flag-tagged FOXN3, HA-tagged ubiquitin, and shRNA targeting β-TrCP were collected 48 hrs post-transfection. f A FOXN3 deletion-mapping assay was carried out in HEK293T cells to identify the specific region accountable for its ubiquitination. The cells transfected with the indicated various mutants of Flag-tagged FOXN3, Myc-tagged β-TrCP, and HA-tagged ubiquitin were collected 48 h post-transfection. g Ubiquitination analysis of WT FOXN3 and its different mutant forms in HEK293T cells to pinpoint the specific site responsible for FOXN3 ubiquitin conjugation. The cells transfected with the indicated various mutants of Flag-tagged FOXN3, Myc-tagged β-TrCP, and HA-tagged ubiquitin were collected 48 h post-transfection. h Immunoblot analysis of the linkage of FOXN3 ubiquitination in HEK293T cells expressing the indicated combinations of Myc-tagged β-TrCP and HA-tagged ubiquitin mutants (K48O or K63O). The cells were collected 48 h post-transfection. i Immunoblot analysis of the linkage of FOXN3 ubiquitination in HEK293T cells expressing the indicated combinations of Myc-tagged β-TrCP, HA-tagged ubiquitin mutant (K63O or K63R) and WT FOXN3 or its K114R mutant. The cells were collected 48 h post-transfection. j Immunoblot analysis was performed in primary pulmonary fibroblasts to assess the impact of WT β-TrCP or its inactive ΔF mutant on the expression of FOXN3. The cells infected with lentivirus expressing WT β-TrCP or its inactive ΔF mutant were collected 48 h post-infection. k Immunoblot analysis was performed in BEAS-2B cells to assess the impact of WT β-TrCP or its inactive ΔF mutant on the expression of WT FOXN3 or its K114R mutant. The indicated combinations were overexpressed in BEAS-2B cells using lentivirus infection, which were collected 48 hrs post-infection. The blotting data (b–k) are representative of two independent experiments and were quantified using ImageJ software.