Neurobiotin tracer coupling was measured by scrape loading in HeLa cells transiently transfected with wild-type Cx35 or with the multiple mutants Cx35-S110A, S276A and Cx35- S110A, S276A, S298ter. Drug treatments were the same as shown in figure 5. Cells transfected with the Cx35 wild-type construct showed a similar pattern of regulation to that seen in the stably-transfected cell line (figure 5). The S110A, S276A double mutant did not show any statistically significant responses to the PKA activator or inhibitor, while the S110A, S276A, S298ter triple mutant showed no response to the PKA activator (Sp) and a small significant reduction in coupling with the PKA inhibitor (Rp). Data are means of 16-24 measurements; error bars are +1 SEM. Significance is displayed as in figure 5.