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[Preprint]. 2024 Oct 2:2024.09.30.615856. [Version 1] doi: 10.1101/2024.09.30.615856

Figure 5. Superior performance of ePKA-KTR1.4 and eERK-KTR1.2 in DRG neurons.

Figure 5.

(A) Graph of C/N ratios of (pink) ePKA-KTR1.2, (purple) ePKA-KTR1.4, and (green) PKA-KTR3 in DRG neurons, at baseline, in response to Fsk, and in response to subsequent addition of H89. ** and **** denote p values <0.01 or <0.0001, respectively. The p values for the changes in PKA-KTR3 C/N ratio were 0.07 and 0.06, respectively. (B) Plot of C/N ratios of ePKA-KTR1.4/tdTomato collected every minute in response to (orange) Fsk alone, (green) Fsk followed later by addition of H89, or (purple) Fsk followed later by addition of PMA. Lines at the top denote the times of chemical addition. (C) Graph of C/N ratios of (green) ERK-KTR1 and (orange) eERK-KTR1.2 in DRG neurons at baseline, in response to PMA, and in response to subsequent addition of H89. *** denote p values <0.001. The p values for the changes in ERK-KTR1 C/N ratio were 0.08 and 0.3, respectively. (D) Plot of C/N ratios of eERK-KTR1.2/emiRFP670 collected every minute in response to (orange) PMA alone, (green) PMA then VX11e, or (purple) PMA then VX11e. Lines at the top denote the times of chemical addition. (E) Representative fluorescence images of DRG co-expressing ePKA-KTR1.4/tdTomato, eERK-KTR1.2/emiRFP670, and ER-mTagBFP2, exposed sequentially to Fsk at t = 3 min, then PMA at t = 23 min, then H89 at t = 45 min, then VX11e at t = 70 min. Images were collected at t = 3, 23, 43, 68, and 93 minutes. Bar, 50 µm. (F) Plot of C/N ratios of (red) ePKA-KTR1.4/tdTomato and (orange) eERK-KTR1.2/emiRFP670 at 1-minute intervals, showing their responses to Fsk, then PMA, then H89, then VX11e. (G) Representative fluorescence images of DRG co-expressing ePKA-KTR1.4/tdTomato, eERK-KTR1.2/emiRFP670, and ER-mTagBFP2, exposed sequentially to PMA at t = 3 min, then Fsk at t = 23 min, then VX11e at t = 45 min, then H89 at t = 70 min. Images were collected at t = 3, 23, 43, 68, and 93 minutes. Bar, 50 µm. (H) Plot of C/N ratios of (red) ePKA-KTR1.4/tdTomato and (orange) eERK-KTR1.2/emiRFP670 at 1-minute intervals, showing their responses to PMA, then Fsk, then VX11e, then H89. Data are from a minimum of three independent biological replicates. p values were calculated by two-way ANOVA.