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. 2020 Apr 16;181(2):346–361.e17. doi: 10.1016/j.cell.2020.03.049

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© 2020 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Figure S4 — Related to Figure 4

A: Phase separated fraction of G3BP1(WT) in comparison to G3BP1(ΔE1) and G3BP1(ΔE2) variants. Phase separation was probed at pH 6 with 75 ng/μl HeLa total RNA and without PEG-20K (mean, SD, fit, n = 20 FOV). B: Quantification of the phase separated fraction of G3BP1 variants in the absence of RNA and PEG-20K, at pH 6 or 7 (mean, SD, fit, n = 20 FOV). C: Left: Quantification of the SG size in live U2O2 G3BP1/2 KO cells transfected with plasmids for expression of mCherry-G3BP1, WT (n = 1013 cells) or ΔE1ΔE2 (n = 1061 cells). Right: representative images of U2OS G3BP1/2 KO cells expressing mCherry-tagged G3BP1(ΔE1ΔE2) or G3BP1(WT), before and 60 min after addition of 1 mM sodium arsenate. Scale bar, 10 μm. D: Immunoblot for expression levels of mCherry-G3BP1 variants in G3BP1/2 KO cells. Tubulin was used as loading control. E: Left: Coomassie (CBB) stained non-native SDS-PAGE showing ~1 μg of purified GFP-labeled G3BP1(WT), G3BP1(S149A), G3BP1(S149E) and G3BP1(S149A/S232A). Right: Immunoblot (IB) against G3BP1 phospho-serine residue 149. From left to right: G3BP1(WT), G3BP1(S149A), dephosphorylated G3BP1(WT) (+PPase). Sample loading was controlled with antibodies against GFP. F: Left, phase separated fraction of G3BP1(WT) phosphorylated (gray) or dephosphorylated (green) with Lambda-phosphatase as a function of pH (mean, SD, fit, n = 16 FOV). Right, phase separated fraction of G3BP1(WT) (gray), G3BP1(S149A) (blue), G3BP1(S149E) (cyan) and G3BP1(S149A/S232A) (green) as a function of pH (mean, SD, fit, n = 25 FOV). Phase separation was tested in the presence of 75 ng/μl of poly(A) RNA and absence of PEG-20K.