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. Author manuscript; available in PMC: 2019 Mar 24.
Published in final edited form as: Nat Cell Biol. 2018 Sep 24;20(10):1215–1225. doi: 10.1038/s41556-018-0200-6

Figure 6. Generating red-shifted single-fluorophore sensors using ddRFP.

Figure 6.

(a) Design and domain structures of the ddRFP-based kinase activity reporters RAB-EKARev and RAB-AKARev. (b) Design and domain structure of the ddRFP-based cAMP reporter RAB-ICUE. (c) Average time-courses (left) and maximum stimulated responses (ΔF/F, right) in HEK293T cells treated with 100 ng/mL EGF. n=70 (RAB-EKARev), 54 (RAB-EKARev[T/A]), and 42 (RAB-EKARev with MEK inhibitor pretreatment [20 μM U0126]) cells. Curves are representative of and bar graphs are pooled from 7, 5, and 3 experiments. ****p<0.0001, F=367.7, DFn=2, DFd=163; one-way ANOVA followed by Dunnet’s test). (d) Average time-courses (left) and maximum stimulated responses (ΔF/F, right) in HeLa cells treated with 50 μM Fsk and 100 μM IBMX (Fsk/IBMX). n=19 (RAB-AKARev), 16, 2 (RAB-AKARev[T/A]), and 22 (RAB-AKARev plus PKI) cells. Curves are representative of and bar graphs are pooled from 3, 2, and 2 experiments. ****p<0.0001, F=138.8, DFn=2, DFd=54; one-way ANOVA followed by Dunnet’s test). (e) Average time-course of the fluorescence response in HEK293T cells expressing RAB-ICUE after Fsk/IBMX treatment. Data are representative of 4 experiments. Curves are normalized with respect to time 0 (F/F0). Solid lines represent the mean, and shaded areas represent SEM. For dot plots shown in c and d, horizontal bars represent mean ± SEM. Maximum responses are calculated as ΔF/F=(Fmax-Fmin)/Fmin. See Supplementary Table 1 for bar graph source data.