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

Figure 1. Tuning NLS strength and KTR size improves PKA-KTR sensitivity and dynamic range.

Figure 1.

(A) Amino acid sequences of PKA-KTR sensor domains, showing the kinase docking site, bNLS, PKA phosphorylation sites (in pink, with asterisk), and NES, with predicted NLS strength scores listed to the right. Bar, 50 μm. (B) Representative fluorescence microscopy images of HEK293 cells expressing the (red) ePKA-KTR1.1, 1.2, 1.3, 1.4, and 1.5 proteins, (blue) H2B-mTagBFP2, and (green) mNeonGreen, grown in the absence of Fsk (-FSK) or after 30 minutes exposure to 30 µM forskolin (+FSK). (C) Bar graph showing C/N ratios of ePKA-KTR1.1, 1.2, 1.3, 1.4, and 1.5/tdCherryproteins (grey) prior to the addition of Fsk or (pink) 30 minutes after the addition of Fsk. (D) C/N ratios of ePKA-KTR1.1, 1.2, 1.3, 1.4, and 1.5/tdCherry (grey) prior to the addition of leptomycin B or (green) after 30 minutes incubation in leptomycin B. (E) Bar graphs comparing the C/N ratios of ePKA-KTR1.2/mCherry (pink) and ePKA-KTR1.2/tdCherry (purple) following 30 minutes incubation in 0, 1, 3, 10, or 30 µM Fsk, or 5 nM leptomycin B (LMB) (F) Bar graphs comparing the C/N ratios of (pink) ePKA-KTR1.4/mCherry and (purple) ePKA-KTR1.4/tdCherry following 30 minutes incubation in 0, 1, 3, 10, or 30 µM Fsk, or 5 nM leptomycin B (LMB). (G) Bar graphs comparing the C/N ratios of ePKA-KTR1.2/mScarlet (gold) or ePKA-KTR1.2/mScarlet/BCR (brown) following 30 minutes incubation in 0, 1, 3, 10, or 30 µM Fsk, or 5 nM leptomycin B (LMB). Data are from a minimum of three independent biological replicates. p values were calculated by two-way ANOVA.