(A) Comparison of the human, mouse and rat Lpar1 promoter sequences with the E-boxes having the CANNTG motif marked and the conserved sequences denoted by asterisks. (B) Putative E-box positions numbered according to their positions and the luciferase construct. (C) Reporter gene assay with promoter constructs containing individually mutated E-boxes. Transient transfection was performed in TR mouse cells (Chun and Jaenisch, 1996) using reporter constructs −761/+225wt, Eb1mut, Eb2mut, Eb3mut, Eb1/2mut, Eb1/3mut, Eb2/3mut and Eb1/2/3mut. Reporter gene activities were normalized to that of the −350/+225 wild-type construct. Each of the mutated E-box sequence is indicated by a dark box (for site Eb1, CATCTG was changed to AGTCTA; for site Eb2, CAGGTG was changed to AGGGTA; and for site Eb3, CAATTG was changed to AGATTA). Error bars indicate S.E. *P < 0.05, ns is non-significant.