Extended Data Fig. 6: 2pFLIM characterizations of cAMPFIREs in HEK cells and as immunoprecipitated proteins. | Nature Methods

Extended Data Fig. 6: 2pFLIM characterizations of cAMPFIREs in HEK cells and as immunoprecipitated proteins.

From: Sensitive genetically encoded sensors for population and subcellular imaging of cAMP in vivo

Extended Data Fig. 6

a, Representative images and lifetime decay curves of cAMPFIRE-M in HEK cells in the presence of the indicated stimuli. Representative of 10 cells from 4 coverslips. b & c, 2pFLIM response traces (b), and NE concentration-response curves in HEK cells fitted by the Hill equation (c), of Epac-SH187 and cAMPFIRE-L, M, and H in HEK cells. Hill coefficients = 0.32, 0.51, 0.79, and 0.84, respectively, for Epac-SH187 and cAMPFIRE-L, M, and H. From low to high concentrations, the n (cells) is: for cAMPFIRE-L, 8, 8, 8, 13, 8, and 8; for cAMPFIRE-M, 6, 8, 8, 10, 9, and 8; for cAMPFIRE-H, 8, 14, 7, 11, and 8; and for Epac-SH187, 9, 8, 11, 4, and 8. Dark lines and shaded areas in panel b, and points and error bars in panel c indicate mean and s.e.m., respectively. d, 2pFLIM response amplitudes of each sensor in HEK cells elicited by 25 µM forskolin and 50 µM IBMX. From left to right, n (cells) = 32, 45, 41, and 48. Bars indicate mean. ***: P < 0.001 for all comparisons, from left to right, P = 1.7 × 10−18, 1.1 × 10−28, and 8.2 × 10−39, dF = 35, 53, and 60, two-tailed Welch test. e, Representative lifetime decay curves of cAMPFIRE-L immunoprecipitated onto Protein A agarose beads in the presence of the indicated cAMP concentrations. Representative of 3 beads. f, 2pFLIM concentration-response curves of cAMPFIRE proteins immunoprecipitated onto Protein A agarose beads in response to cAMP. Points and error bars indicated mean and s.e.m., respectively. For cAMPFIRE-L, -M, and -H, respectively, n = 3, 4 and 4 beads; Hill coefficients = 0.65, 0.68, and 0.82.

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