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. 2002 Nov 1;99(23):15142–15147. doi: 10.1073/pnas.232565699

Fig 3.

Fig 3.

Time-lapse measurements of induced Bla enzymatic activity in single primary cortical neurons. (A) A neuron expressing the KID/KIX-BEAM proteins was identified by detecting fluorescence emission characteristic of the coexpressed DsRed2 red fluorescent protein. (B) Pseudocolor images of the CCF-2 R460/530 captured during the time-lapse imaging experiment for the KID/KIX-BEAM-expressing neuron identified in A. The neuron was treated with 10 μM forskolin (fsk) plus 300 μM CPT-cAMP 360 s after the start of the experiment. Bla enzymatic activity increased significantly after treatment with PKA agonists (t = 880 s) and continued to increase over the course of the experiment (t = 1,500 s). (C) CCF-2 R460/530 plots as a function of time for three neurons expressing the KID/KIX-BEAM proteins. A sustained increase in the slope, dR(t)460/530/dt, was observed in the presence of PKA agonists for the period of time indicated by the black bar above the plots. (D) Plots of relative Bla enzymatic activity, kBla(t), for the three neurons whose R460/530 plots as a function of time are shown in C. The magnitude of the relative increase in Bla enzymatic activity for these three neurons after stimulation with PKA agonists was 3.25 ± 0.16-, 3.45 ± 0.06-, and 4.49 ± 0.12-fold, respectively. (E) Average relative increase in Bla enzymatic activity for 49 neurons expressing the KID/KIX-BEAM proteins. An induced 2.71 ± 0.08-fold change was observed after treatment with forskolin and CPT-cAMP. (F) The average of 53 individual kbla(t) plots for neurons expressing the M1-KID/KIX-BEAM proteins showed no change in relative enzymatic rate after treatment with forskolin and CPT-cAMP.