Fig. 2.

Endogenous NPR-A is down-regulated by ANP in immortalized human cervical HeLa cells. A and B, Cells were incubated with 200 nm ANP for the indicated periods of time at 37 C. Cycloheximide (10 μg/ml) was also added to the medium to block protein synthesis. A, Membranes were prepared and assayed for guanylyl cyclase activity after basal, ANP, or Triton X-100 stimulation. Activity levels were plotted as a function of time in the presence of ANP. Data points are represented as mean ± sem, where n = 3. B, In a parallel experiment, NPR-A was immunoprecipitated and detected by immunoblot. NPR-A isolated from 293T cells stably expressing NPR-A was used as a positive control. A synthetic NPR-A blocking peptide was used as a negative control to block NPR-A antibody/antigen binding. NPR-A protein levels from four independent immunoblots were quantitated, normalized, and presented in graphical form as mean ± sem, where n = 4. Statistical significance was determined by a paired t test, where *, P < 0.005. C and D, Cells were transfected with siRNA against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or NPR-A. C, NPR-A was immunoprecipitated and detected by immunoblot to verify the NPR-A knock down. D, Membranes were prepared and assayed for guanylyl cyclase activity after a 3-min basal, ANP, or Triton X-100 stimulation. Data points are represented as mean ± sem, where n = 3.