Topology of signaling networks studied. Two general types of network motifs consisting of covalently modified cycles were studied: (A) the vertical case where the n-th cycle in the right hand cascade is inhibited and (B) the lateral case where the n-th single-cycle cascade is inhibited. (C) The n = 3 network consisted of exactly 3 cycles and was the simplest form of both the vertical and lateral case. (D) An extended n = 3 network was also studied where a fourth cycle activated by Y2* was added to the left-most cascade. In all networks, Y1* served as the upstream activator and cycle 2 and cycle 3 were always in distinct cascades. No additional regulatory connections were included in any network. Off-target effects in cycle 2 were monitored by measuring the steady state concentrations of Y2 and Y2* as the concentration of an inhibitory drug that specifically targeted Y
* was increased.