Figure 3.

TanFe (transcellular activator of nuclear FOXF1 expression) disrupts FOXF1 (Forkhead Box F1)–HECTD1 protein–protein interactions. (A) Immunoprecipitation (IP) shows that HECTD1 interacts with endogenous FOXF1 protein. IP was performed with cell lysates from Mouse Fetal Lung Mesenchyme-91 U (MFLM-91U) cells using the HECTD1 antibody or IgG control. Protein samples were pooled from five independent cell cultures. (B) IP shows that FOXF1 interacts with HECTD1 protein. IP was performed with cell lysates from MFLM-91 U cells stably expressing His-Flag–tagged mouse FOXF1. Cells were treated with 20 μM of TanFe or vehicle (Dimethyl sulfoxide) for 24 hours. FOXF1-interacting proteins were purified using anti-Flag M2 agarose beads and Talon metal affinity resin and were analyzed by Western blot. (C) The in vitro ubiquitination assay shows that HECTD1 ubiquitinates the FOXF1 protein. HA-HECTD1 or HA-HECTD1 C2579G mutants were expressed in Human Embryonic Kidney (HEK) 293T cells, purified using HA magnetic beads, and eluted using HA peptide. The ubiquitination assay was performed in the presence of FLAG-FOXF1 immobilized magnetic beads and the purified HA-HECTD1 proteins. Binding of biotinylated Ub to FLAG-FOXF1 protein was detected by Western blot using Horseradish peroxidase (HRP) streptavidin. (D) Western blot shows that increasing TanFe concentrations interfere with binding of purified HECTD1 and FOXF1 proteins. Endogenous HECTD1 was immunoprecipitated from MFLM-91 U cells and incubated with the purified FLAG-FOXF1 protein immobilized on agarose beads. Protein samples were pooled from three cell cultures. Ub = ubiquitin; UBE3A = Ubiquitin protein ligase E3A.