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. 2012 Mar 1;125(5):1106–1117. doi: 10.1242/jcs.089086

Fig. 3.

Fig. 3.

FRET analysis of PKA activity in the sub-membrane region and bulk cytosol of epithelial cells. (A–D) Analysis of the membrane-targeted version of AKAR3, mpAKAR3. (E–H) Analysis of cytosolic AKAR3. Distribution of mpAKAR3 (A) and AKAR3 (E) reporters respectively, expressed in HBE, CFBE and CFBE/sNHERF1 cells. (B,F) Representative kinetics of PKA-mediated phosphorylation recorded in the cells shown in A and E, respectively, upon stimulation with 40 μM Frsk and 100 μM IBMX. R is the normalized 545 nm/480 nm value calculated at each acquisition time point. Black line, dotted gray line and dotted black line represent kinetics recorded respectively in HBE, CFBE and CFBE/sNHERF1 both in the bulk cytosol and at the plasma membrane. (C,G) Intensity-modulated pseudocolor images of the cells shown in A and E, respectively. For each cell the 545/480 nm emission ratio values before (time=0 seconds) and 1500 seconds after the addition of 40 μM Frsk and 100 μM IBMX are shown. (D,H) Summary of all experiments performed in B and F, respectively, showing FRET changes recorded in HBE, CFBE and CFBE/sNHERF1 cells expressing the mpAKAR3 or AKAR3 sensors upon stimulation with 40 μM Frsk and 100 μM IBMX. Data represent means ± s.e. Scale bars: 10 μm. ***P<0.001; *P<0.05.