FOXC2 p.H395N variant decreases protein stability.

Time course stability analysis of FOXC2 (A) and PITX2 (C) coding variants found in PCG patients was carried out by transient expression in HEK-293T cells. Transfected cells were treated with the protein synthesis inhibitor cycloheximide and the different recombinant proteins were detected by Western immunoblot using an anti-myc monoclonal antibody (Santa Cruz) at the indicated time points, as explained in Materials and Methods. Relative amounts of FOXC2 are expressed as a percentage of levels at time 0 h. The rate of decay and half-lives of the recombinant FOXC2 (B) and PITX2 (D) coding variants at the indicated time-points were determined from linear regression analysis as described in the Materials and Methods. Error bars correspond to the SD of three independent experiments carried out in triplicate. Asterisks indicate statistical significance compared to the control: p<0.01 (**). Two-way ANOVA followed by Tukey multiple-comparison test.