Functional assessment of the identified LHX4 variants. (A–D) Transactivation of the POU1F1, GH, and PRL proximal promoters by LHX4 variants. CHO cells were transiently cotransfected with 30 ng of cDNA expression vectors for wild-type (WT) or mutated LHX4 and 100 ng of a luciferase reporter gene under the control of the POU1F1, GH, or PRL promoter. Promoter activity was assessed by measuring luciferase activity 24 hours after transfection. For negative and half-dose controls, the total amount of plasmid was adjusted to 30 ng with the empty expression vector. Results are expressed in relative light units normalized by the total amount of protein (in µg) and adjusted to WT activity. The data are the mean ± SD of 3 independent experiments, each performed in triplicate. (A–C) P < 0.01 compared with WT (*). (D) P < 0.01 compared with 1/2 mock, 1/2 WT (*). (E) Coimmunoprecipitation of HA-LHX4 and FLAG-ISL2. HEK293T whole-cell lysates were immunoprecipitated with anti-FLAG antibody (ISL2). LHX4 was revealed using anti-HA antibody. (F) Coimmunoprecipitation of HA-LHX4 and FLAG-LHX3. HEK293T whole-cell lysates were immunoprecipitated with anti-FLAG antibody (LHX3). LHX4 was revealed using anti-LHX4 antibody (Santa Cruz Biotechnology). Input represents 10% of total lysates.