Figure 4.

a, Impaired activation of Gal4-SOX3(A₍₇₎240∧241 ins) (labeled “SOX3 (22 ala)”) at SOX binding sites. Increasing concentrations of Gal4-SOX3 (labeled “SOX3 (15 ala)”) and Gal4-SOX3(A₍₇₎240∧241 ins) (10 ng, 100 ng, 500 ng, and 1,000 ng) were cotransfected with the HESX1 promoter (Hesx1-PP) luciferase reporter. Gal4-SOX3 led to a dose-dependent activation of the HESX1 promoter (a 6-fold activation at the highest concentration tested), whereas Gal4-SOX3(A₍₇₎240∧241 ins) was associated with impaired activation (a 3-fold activation at the highest concentration tested). b, Impaired activation of Gal4-SOX3(A₍₇₎240∧241 ins) (labeled “SOX3 (22 ala)”) at the SV40 promoter. Increasing concentrations of Gal4-SOX3 (labeled “SOX3 (15 ala)”) and Gal4-SOX3(A₍₇₎240∧241 ins) (10 ng, 100 ng, 500 ng, and 1,000 ng) were cotransfected with the SV40 promoter reporter construct. Gal4-SOX3 led to a dose-dependent activation of the SV40 promoter (a 3.3-fold activation at the highest concentration tested), whereas Gal4-SOX3(A₍₇₎240∧241 ins) was associated with impaired activation (a 2-fold activation at the highest concentration tested). a and b, One asterisk (*) indicates P<.05, two asterisks (**) indicate P<.001, and three asterisks (***) indicate P<.0001. The results represent the means of three independent experiments, each performed in triplicate. c, Gal4-SOX3(A₍₇₎240∧241 ins) (labeled “SOX3 (22 ala)”) binds to SOX binding sites in a manner that is similar to that of wild-type SOX3 (labeled “SOX3 (15 ala)”). Identical amounts of in vitro translated wild-type SOX3 or SOX3(A₍₇₎240∧241 ins) (10 μl) were added to the consensus SOX DNA-binding site (lanes 2 and 4). Duplicate experiments—but with the addition of 15 pmol of cold probe, showing specific loss of DNA binding—are shown in lanes 3 and 5. Lane 1 shows the effect of free probe in the absence of protein. Western blot analysis showed equivalent expression levels of the constructs (data not shown). d, Impaired nuclear localization in Gal4-SOX3(A₍₇₎240∧241) (labeled “SOX3 (22 ala)”). CHO cells were transfected with 50 ng of the respective plasmid construct, as indicated. Nuclei in the cells were counterstained with DAPI. Panels i–vi, wild-type SOX3 (labeled “SOX3 (15 ala)”), using either α-GAL4 (panels i–iii) or α-cSOX3 (panels iv–vi) antibodies; panels vii–xii, Gal4-SOX3(A₍₇₎240∧241) (labeled “SOX3 (22 ala)”), using either α-GAL4 (panels vii–ix) or α-cSOX3 (panels x–xii) antibodies.