FIGURE 11.

Cellular stability and transcriptional activities of human SRY and variants. A, verification of expression of full-length SRY and variants in CH34 cell model. B, Western blot of variants used in standard and “double-rescued” experiments. Tethering of exogenous NLS (+NLS, green labels) increases molecular weight and shifts specific bands. The decreased protein accumulation observed for W70L and W70A (lanes 2 and 3, respectively) is circumvented by the use of a proteasome inhibitor (lanes 6 and 8, +MG132). C, diagram of full-length human SRY plasmids used in these studies; position of the exogenous NLS (for double-rescue experiments) is highlighted in sequences (green) below diagram. D, cycloheximide assay indicates that proteolysis is enhanced for the nonaromatic variants W70L and W70A (red and gray lines, respectively); however, W70F and W70Y (blue and purple lines, respectively) exhibit a similar cellular lifetime compared with WT (black line). E, relationship between Sox9 expression and SRY (WT and variant) at two doses indicated by solid (high concentration) and white (low concentration) transfected HA-SRY expression plasmid. qPCR data were obtained under standard conditions (left in black) and double-rescued (right in green) in the presence of MG132 with N-terminally fused SV40 nuclear localization signal (+NLS). **, p < 0.01; ns, not significant.