Skip to main content
. Author manuscript; available in PMC: 2022 Aug 18.
Published in final edited form as: J Am Chem Soc. 2021 Aug 4;143(32):12675–12687. doi: 10.1021/jacs.1c05386

Figure 7|. Phosphorylation at S61 by PKC in vitro dissociates NBDY-RNA liquid droplets.

Figure 7|

(a) NBDY-polyU coacervate dissolution by PKC activity. Top row: transmission brightfield images. Bottom row: TMR-NBDY fluorescence at the same time points. Buffer: 25 mM HEPES, pH 7.4, 20 μM CaCl2, 50 mM NaCl, 10 mM DTT, 100mM MgCl2, 0.5 mg/ml phosphatidylserine, 100 μM DTT, 0.5 mM sodium azide, 0.5% w/v Tryptone, 10% PEG3350 at 30 °C. 0.4% w/v polyU, 200 ng PKCα and 200 μM NBDY were used. Scale bars, 10 μm. (b) Solution turbidity as a function of time for purified wild-type (red), S61A NBDY (blue) or S61D NBDY (green) protein in the presence of PKC. Error bars represent the standard deviation of three independent trials. Buffer: 25 mM HEPES, pH 7.4, 20 μM CaCl2, 50 mM NaCl, 10 mM DTT, 100mM MgCl2, 0.5 mg/ml phosphatidylserine, 100 μM DTT, 0.5 mM sodium azide, 0.5% w/v Tryptone, 10% PEG3350 at 30 °C. 0.4% w/v polyU, 200 ng PKCα and 200 μM wild-type or mutant NBDY were used.