Binding assays of NKX3.1 binding sites with nuclear extracts. A. Locations and sequences of five potential NKX3.1 binding sites (NBSs) upstream of the PCAN1 gene. B. EMSA was performed to assay the binding activities of the five NBSs with the nuclear extracts from LNCaP cells. 1–5: labelled NBS1~NBS5 with LNCaP nuclear extracts respectively. A DNA-protein binding complex was formed when the labelled probe of NBS1 (lane 1) or NBS3 (lane 3) was reacted with LNCaP nuclear extracts. C and D. Competition binding and antibody blocking were used to detect the specific binding of the NBS1 (C) or NBS3 (D) with NKX3.1. Labelled NBS1 or NBS3 without nuclear extracts was used as the control (lane1); a specific DNA-protein complex was formed when labelled NBS1 or NBS3 was reacted with LNCaP nuclear extracts (lane 2). Competition binding was performed in the presence of a 250-fold molar excess of unlabelled NBS1 or NBS3 (lane 3), unlabelled mutant NBS1 or NBS3 (lane 4). The binding activity of probe NBS1 or NBS3 to nuclear extracts could be blocked by anti-NKX3.1 antibody (lane5).