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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 4. Construction of ExRai-CKAR and ExRai-AktAR based on a generalized design.

Figure 4.

(a) Domain structures of ExRai-CKAR (top) and ExRai-AktAR (bottom). (b) Average time-courses (left) and maximum stimulated responses (ΔR/R, right top) of HeLa cells treated with 100 ng/mL PMA. n=26 (ExRai-CKAR), 17 (ExRai-CKAR[T/A]), and 11 (ExRai-CKAR with PKC inhibitor pretreatment [10 μM Gö6983]) cells. Curves are representative of and bar graphs are pooled from 5, 3, and 3 experiments. (****p<0.0001, F=351.9, DFn=2, DFd=51; one-way ANOVA followed by Dunnet’s multiple comparisons test). Representative pseudocolor images show the ExRai-CKAR excitation ratio in HeLa cells before and after stimulation. Warmer colors correspond to higher excitation ratios. (c) Average time-courses (left) and maximum stimulated responses (ΔR/R, right top) of NIH3T3 cells treated with 50 ng/mL PDGF. n=15 (ExRai-AktAR), 16 (ExRai-AktAR[T/A]), and 7 (ExRai-AktAR with Akt inhibitor pretreatment [50 μM10-DEBC]) cells. Curves and bar graphs are pooled from 12, 4, and 5 experiments. (****p<0.0001, F=445.3, DFn=2, DFd=33; one-way ANOVA followed by Dunnet’s multiple comparisons test). Representative pseudocolor images show the ExRai-AktAR excitation ratio in NIH3T3 cells before (middle) and after (bottom) stimulation. Warmer colors correspond to higher excitation ratios. Scale bars, 10 μm. Curves are plotted as 480/380 excitation ratio normalized with respect to time 0 (R/R0). Solid lines represent the mean; shaded areas, SEM. For dot plots in b and c, horizontal bars represent mean ± SEM. Maximum responses are calculated as ΔR/R=(Rmax-Rmin)/Rmin. See Supplementary Table 1 for bar graph source data.