IGF-1 regulates secreted TIMP levels. DU145 cells were pre-treated for 1 hour with either the PI3-K inhibitor wortmannin or the MEK inhibitor PD98059, then treated with IGF-1 for 24 hours in the same conditions of inhibitor use. The control consisted of adding a similar amount of the vehicle (1× PBS for IGF-1, DMSO for wortmannin or PD98059) for each addition in the test conditions. Conditioned media from these treatments were concentrated and probed with (A) anti-TIMP-2 antibody or (B) anti-TIMP-1 antibody. The amount of protein in each lane is normalized to cell number. Blots are representative of three separate experiments in which a similar trend was observed.