Figure 6. Functional impairment of Foxn1 is dependent on the location and type of mutation.

(A–C) Transient transfection assays in HEK293T cells were performed with expression vectors for WT Foxn1 or Foxn1 constructs harboring the indicated mutations that matched the FOXN1 mutations identified in patients. The mutations were divided into those identified in Pt. 1 (A) (Foxn1⁹³³, Foxn1¹⁰⁸⁹) and Pt. 2 (B) (Foxn1¹²⁸⁸, Foxn1¹⁴⁶⁵) as well as the indicated controls. Forty-eight hours after transfection, the cells were lysed, and the proteins were extracted and resolved by SDS-PAGE. Western blotting was performed with antibodies against Foxn1, followed by antibodies detecting GAPDH, which was used as a loading control. Blots are representative of 4 independent experiments. (C and D) HEK293T cells were transfected with the indicated constructs along with a Psmb11 luciferase reporter construct and a β-gal vector. The mutations (mFoxn1) were grouped for those identified in Pt. 1 (C) (Foxn1⁹³³, Foxn1¹⁰⁸⁹) and Pt. 2 (D) (Foxn1¹²⁸⁸, Foxn1¹⁴⁶⁵) along with controls (D). Forty-eight hours after transfection, the cells were harvested, and luciferase activity was measured. The luciferase activity was normalized to β-gal, which was used as an internal control. Data are representative of triplicate samples from each group using 3 independent transfections per group. P values were determined using a standard 1-way ANOVA.