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. 2013 Oct 15;126(20):4614–4626. doi: 10.1242/jcs.122952

Fig. 4.

Fig. 4.

Non-adaptive response of ACA in CM1234 cells. (A) Differentiated WT-CAR1 (n = 5) and CM1234 (n = 5) cells were stimulated at 22°C with 10 µM cAMP. At the times indicated, lysates were prepared and ACA activity measured for 1 minute. Owing to the variability of total cAMP produced among the assays, background counts were subtracted from raw values and activation data (with s.e.m.) were normalized to maximal activity within each experiment. P<0.0001 by ANOVA. (B) Differentiated WT-CAR1 (n = 2) and CM1234 (n = 2) cells were stimulated at 22°C with 5 µM 2′deoxy-cAMP in the presence of 10 mM DTT. Total cAMP (with s.e.m.) was measured at the times indicated. A plateau in cAMP accumulation for WT-CAR1 cells corresponds to ACA adaptation observed at ∼1 minute (see Fig. 4A). P<0.0001 by ANOVA. C. Differentiated WT-CAR1 and CM1234 were stimulated at 22°C with varying concentrations of cAMP, as indicated. To accurately measure dose responses, a single, differentiated cell population was stimulated with the indicated concentration of cAMP, with a 20 second time lag between separate assays (see the Materials and Methods). Experiments were repeated at least twice with duplicates for each time point. Values indicate means ± s.d. (D) WT-CAR1 and CM1234 cells were differentiated, and stimulated at 4°C with 100 nM cAMP in the absence or presence of 10 mM DTT, with a 20 second time lag between each assay. At the times indicated, lysates were prepared and ACA activity assayed for 1 minute at 22°C. (E) WT-CAR1 and CM1234 cells were differentiated and stimulated at 4°C with different concentrations of cAMP in the presence of 10 mM DTT, with a 20 second time lag between each stimulation. At the times indicated, lysates were prepared and ACA activity assayed for 1 minute at 22°C. (F) WT-CAR1 (circles) or CM1234 (boxes) cells were differentiated and stimulated at 4°C in the presence of DTT with the indicated concentrations of cAMP. After 1 minute, cells were lysed and assayed for ACA activity for 1 minute. Data from two experiments (open/closed symbols) are plotted with an averaged curve fit between the points.