FIGURE 3.

Absence of an S1P effect on cAMP responses in AC7^-/- BMDMs. A, BMDM cells were isolated from 6-week-old female mice of the same litter. The genotype of the BMDMs was determined by PCR. B-F, freshly isolated BMDMs were infected with retrovirus carrying the CAMYEL sensor. cAMP responses were measured using the BRET assay. B, wild type BMDMs were treated with buffer alone, 100 ng/ml pertussis toxin (PTx) for 20 h, or 10 μm thapsigargin and 2 mm EGTA (TG/EGTA) for 2 min as indicated and stimulated with sequential addition of 4 nm ISO at time 0 followed by 4 nm S1P at 120 s. C-E, three genotypes of BMDM cells were treated with sequential addition of 4 nm ISO at time 0 followed by 4 nm S1P (C), 50 nm C5a (D), or 500 nm UDP (E) at 120 s as indicated by arrows. F, three genotypes of BMDM cells were stimulated with 160 nm ISO at time 0. Representative error bars for selected traces are the standard deviation of results from three experiments.