FIG. 5.

Pitx-1 interacts directly with c-Jun. (A) EMSA was performed using GST/Pitx-1 and hc-Jun. −300/−259 was used as the probe. Each combination of GST/Pitx-1 (500 ng/lane), hc-Jun (0.1 fpu/lane), α-Pitx-1 antibody, and/or α-c-Jun antibody was incubated with probe. GST (500 ng/lane) was used as a negative control. *, new protein-DNA complex in the presence of both Pitx-1 and c-Jun. (B) GST pull-down analysis using hc-Jun (0.1 fpu/lane) and GST/Pitx-1 (1 μg/lane) was performed as described in Materials and Methods. GST (1 μg/lane) was used as a negative control. For the 10% input lane, 0.01 fpu of hc-Jun was loaded. (C) Luc reporter (pT109) containing 5xUAS was cotransfected into CV-1 cells with either Pitx-1 or empty expression vector and the indicated GAL4 DBD fusion constructs. Data are plotted as induction by Pitx-1 over the empty expression vector control response for each GAL4 DBD fusion construct. Data are presented as the mean ± SEM of three independent experiments. *, P < 0.05 versus GAL4 empty vector. (D) Co-IP analysis was performed using nuclear extracts (245 μg/lane) from LβT2 gonadotrope cells treated with 100 nM GnRH agonist for 1 h. IP was performed by incubating nuclear extracts with preimmune IgG, α-c-Jun antibody, or α-Pitx-1 antibody, followed by Western blot analysis with α-Pitx-1 antibody. For the input lanes, 10 μg of each nuclear extract with or without GnRH agonist treatment was loaded. IgG, preimmune IgG.