GC-box "a" is essential for TRPV1 P2-promoter activation in DRG neurons and NGF-treated PC12 cells. Comparison of P2-promoter activity in DRG neurons + NGF (A) or +/- NGF-treated PC12 cells (B) directed by: empty pGL3 reporter plasmid (pGL-E); control reporter plasmid (0.4 kb); 0.4 kb reporter with deletion of GC-box "a" (Del-a); 0.4 kb reporter with deletion of GC-box "b" (Del-b) or deletion of both GC-box "a & b". Deletion of GC-box "a" resulted in a complete loss of promoter activity when compared with the (0.4 kb) P2-promoter control in DRG neurons and NGF treated PC12 cells. Deletion of GC-box "b" directed a trend to decrease promoter activity in DRG neurons and NGF treated PC12 cells. Concurrent loss of GC-box a & b resulted in the lowest measurable promoter activity. When identical experiments were repeated under conditions of human Sp1 cDNA (A,B) over-expression, P2-promoter activity continued to be lost following deletion of GC-box "a" or GC-box "a & b". Loss of GC-box "b" under conditions of Sp1 (A,B) over-expression showed a small decrease of P2-promoter activity that attained significance in NGF treated PC12 cells. Diagram (left) indicates location of GC-box deletions and start site of transcription for P2-promoter expressing firefly luciferase (Luc). Error bars SEM (n = 3) quadruplicate measures. Significant differences: ANOVA (***) p <0.001; (*) p < 0.05.