Journal: bioRxiv

Article Title: Molecular insights into the effect of alkanediols on FUS liquid-liquid phase separation

doi: 10.1101/2022.05.05.490812

Figure Lengend Snippet: a) DIC micrographs of 5 μM FUS full-length without (top) and with (bottom) polyadenylic acid (0.27 mg/ml polyA) after treatment with 5% w/v premixed solutions of aliphatic alcohols and addition of TEV protease. Scale bars represent 50 μm. b) Phase separation of 5 μM FUS full-length without (left) and with (right) polyA RNA over the course of 5 hours as measured by light scattering at 600 nm. Error bars represent standard deviation of three replicates. c) DIC micrographs of 100 μM FUS RGG3 (453-507) after the addition of 0.142 mg/ml of polyA RNA in different LLPS-modifying buffers. d) Saturation concentration of 100 μM FUS RGG3 in the presence of polyA RNA (0.142 mg/ml) diluted in 50 mM MES pH 5.5, 150 mM NaCl, 5% w/v co-solvent. Data are plotted as mean ± s.d. of n=3 technical replicates. e) The effect of different alkanediols at 5% w/v, pH 5.5 on phase separation of 100 μM FUS RGG3 in the presence of RNA over time as tracked by light scattering at 600 nm. Error bars represent standard deviation of three replicates.

Article Snippet: FUS SYQG LC containing a TEV cleavable N-terminal histidine tag (RP1B FUS LC, AddGene #127192), full-length FUS with an N-terminal histidine tag and maltose-binding protein fusion (pTHMT FUS 1-526, AddGene #98651), and FUS RGG3 with N-terminal histidine tag and MBP fusion (pTHMT FUS RGG3 (453-507)) were expressed in Escherichia coli and grown at 37°C to an OD of 0.60-0.90 before induction with 1 mM IPTG for 4 hours.

Techniques: Standard Deviation, Concentration Assay

Journal: bioRxiv

Article Title: Molecular insights into the effect of alkanediols on FUS liquid-liquid phase separation

doi: 10.1101/2022.05.05.490812

Figure Lengend Snippet: Droplets were made in 50 mM MES pH 5.5, 150 mM NaCl, 5% co-solvent, 100 μM FUS RGG3, 0.142 mg/ml polyadenylic acid (poly A) RNA and were imaged after 1 hour of incubation in room temperature. Scale bars represent 50 μm.

Article Snippet: FUS SYQG LC containing a TEV cleavable N-terminal histidine tag (RP1B FUS LC, AddGene #127192), full-length FUS with an N-terminal histidine tag and maltose-binding protein fusion (pTHMT FUS 1-526, AddGene #98651), and FUS RGG3 with N-terminal histidine tag and MBP fusion (pTHMT FUS RGG3 (453-507)) were expressed in Escherichia coli and grown at 37°C to an OD of 0.60-0.90 before induction with 1 mM IPTG for 4 hours.

Techniques: Incubation

Journal: bioRxiv

Article Title: Molecular insights into the effect of alkanediols on FUS liquid-liquid phase separation

doi: 10.1101/2022.05.05.490812

Figure Lengend Snippet: a) 1 H- 15 N heteronuclear single quantum coherence (HSQC) spectrum of 100 μM FUS RGG3 with (blue) and without (red) 5% 1,6-hexanediol. b) Chemical shift deviations of 1 H FUS RGG3 and 15 N FUS RGG3 (c,d) binned by residue type. Individual chemical shifts are plotted as blue marks. Bar plots represent mean and s.d. among all chemical shifts of each residue type.

Article Snippet: FUS SYQG LC containing a TEV cleavable N-terminal histidine tag (RP1B FUS LC, AddGene #127192), full-length FUS with an N-terminal histidine tag and maltose-binding protein fusion (pTHMT FUS 1-526, AddGene #98651), and FUS RGG3 with N-terminal histidine tag and MBP fusion (pTHMT FUS RGG3 (453-507)) were expressed in Escherichia coli and grown at 37°C to an OD of 0.60-0.90 before induction with 1 mM IPTG for 4 hours.

Techniques: