Figure 3.

A, PC3 cells were transfected with increasing doses of NKX3.1-expressing plasmid. Total RNA was collected and subjected to real-time PCR using primers for VEGF-C and 36B4 (housekeeping gene). The data presented is the mean of three individual experiments. B, PC3 were transfected with the NKX3.1-expressing plasmid as mentioned in Fig. 2E. Cell lysates were subjected to Western blotting using antibody for NKX3.1. C and D, recruitment of NKX3.1 to VEGF-C promoter. C, ChIP assay. Cross-linked chromatin-protein complexes were isolated from LNCaP cells cultured in 10% normal serum (NS) and rum (CS, androgen deprived). Immunoprecipitation of chromatin fragments was carried out with the NKX3.1 as well as rabbit IgG (control) antibody. After reverse cross-linking, DNA fragments were isolated and the NKX3.1 binding region on the VEGF-C promoter was amplified by PCR with the VEGF-C promoter–specific primers. An amplified PCR product was detected when immunoprecipitation was carried out with the NKX3.1 antibody. D, nuclear extracts from LNCaP cells were cultured in 10% normal serum and CS, and were subjected to Western blotting using antibody against NKX3.1.