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. 2021 Mar 2;10:e64252. doi: 10.7554/eLife.64252

Figure 2. Specific RNAs induce formation of dynamic mesh-like condensates in vitro.

(A) Representative confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the absence or presence of the indicated in vitro transcribed RNAs after 16 hr of incubation. Scale bar, 2 µm. Five percent dextran was added into the phase separation buffer as crowding agent in all experiments. (B) Fluorescence recovery after photobleaching of mGFP-FUS-TIS (10 µM) mixed with CD47 3′UTR (50 nM) after 2 hr of incubation. Scale bar, 1 µm. (C) Same as (A), but in the presence of different RNA concentrations. (D) Same as (A), but additional RNAs are shown.

Figure 2.

Figure 2—figure supplement 1. Specific RNAs induce mesh-like condensates in vitro.

Figure 2—figure supplement 1.

(A) Schematic of the FUS-TIS chimeric protein. (B) SDS-PAGE of the purified FUS-TIS protein. FUS-TIS was tagged with His-MBP-mGFP at the N-terminus and with Strep-tag II at the C-terminus and purified from E. coli. mGFP: monomeric GFP. (C) Size exclusion chromatography of FUS-TIS protein. (D) Phase separation of purified mGFP-FUS-TIS protein at the indicated concentrations was induced by reducing the salt concentration from 600 mM to 150 mM through dilution; shown is a 2 hr time point. The His-MBP tag was cleaved off before the phase separation experiment using TEV protease. Scale bar, 10 µm. (E) Snapshots showing a fusion event of mGFP-FUS-TIS condensates at 2 hr. (F) Fluorescence recovery after photobleaching of mGFP-FUS-TIS (10 µM) mixed with CD47 3′UTR (50 nM) after 16 hr of incubation. Scale bar, 2 µm. (G) Representative confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the absence or presence of the indicated in vitro transcribed RNAs after 16 hr of incubation. Scale bar, 2 µm.
Figure 2—figure supplement 2. Specific RNAs induce mesh-like condensates in vitro.

Figure 2—figure supplement 2.

(A–D) Representative confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the absence or presence of the indicated in vitro transcribed RNAs after 2 hr of incubation. Scale bar, 2 µm.