Binding activities of various combinations of MEIS1, PBX1B, and PBX2 to the TME and their effect on transcriptional activities of the PF4 promoter. (A) HEL NE (nuclear extract) and MEIS1, PBX1B, and PBX2 prepared by in vitro translation were used in EMSA. Supershift assays were also performed with antibodies to MEIS1, PBX1B, and PBX2. (B) EMSA was performed with in vitro translated proteins. The TME and 2 mutant TME probes were used. Mut-2 probe is the same sequence as Mut-2 described in Figure 2A. The MutTGACAG probe is the mutant TME probe in which 2 Meis1 binding sites are disrupted (TGACAG to AGCGAT). (C) The plasmids for the expression of MEIS1, PBX1B, and PBX2 were transfected into HepG2 cells with PF4luc or PF4mut reporter-plasmids. PF4luc contains 1.1 kb PF4 promoter in front of the luciferase reporter gene. PF4mut contains mutations in the Meis1 binding sites in the TME. Error bars indicate SDs.