Fig. 1. NAD+/ATP induces phase separation of TIR-only proteins in vitro.
a, Images showing NAD+-induced liquid-like droplets of RPP1-TIR–GFP. NAD+ (1 mM) was incubated with 25 μM RPP1-TIR protein in the presence of 5% polyethylene glycol (PEG) 3,350 at 25 °C for 30 min. Scale bar, 10 μm. b, Dynamics analysis of RPP1-TIR–GFP droplets by FRAP. The photobleaching pulse ends at 0 s. Scale bar, 1 μm. c, Quantification of the RPP1-TIR–GFP droplets in FRAP assays. Data are mean ± s.d. (n = 3 biologically independent samples). d, Images showing ATP-induced droplets of RPP1-TIR–GFP; 1 mM ATP was incubated with 25 μM RPP1-TIR protein in the presence of 5% PEG 3,350 at 25 °C for 30 min. Scale bars, 10 μm. e,f, NAD+ induces liquid-to-solid transition of the RBA1 and TX14 TIR domain proteins. NAD+ was incubated with 10 μM RBA1 or TX14 at 25 °C for 30 min. Scale bars, 20 μm. a,d,e,f, Experiments were repeated at least three times with similar results.