HES1 affects FANCF and FANCG protein stability. (A) 293T cells were cotransfected with FA gene expression plasmids as indicated with either empty, or BACE1 or HES1 coding vectors. Both protein and RNA were extracted and subjected to either SDS-PAGE (left panel) or RT-PCR (right panel) of each gene as indicated. Endogenous tubulin protein and GAPDH RNA expression were used as internal controls. (B) Dose-dependent effect of HES1 on FANCF protein stability. 293T cells were cotransfected with FANCF expression plasmids along with increasing amounts of HES1 coding vector as indicated (μg). The total amount of transfected plasmid was equalized for all strategies with control empty vectors. (C,D) 293T cells cotransfected with FANCF or FANCG along with either empty, or BACE1 or HES1 expression plasmids were treated with the proteasome inhibitor ALLN (50 μM) for 16 hours following transfection. The FANCA expression plasmid was used as a positive control for FANCG stabilization.