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. 2021 Nov 30;12:6984. doi: 10.1038/s41467-021-27306-4

Fig. 8. RAD23B mediates SIPAN formation.

Fig. 8

a Analysis of domains and intrinsically disordered regions of RAD23 proteins. Prediction of order/disorder propensity of human RAD23B (left panel, uniprot #P54727) and yeast RAD23 (right panel, uniprot #P32628) based on their protein sequences. Disorder scores were calculated using PONDR-FIT (green), VSL2B (blue), and VLXT (magenta). b, c RAD23B lacking critical domains failed to assemble in SIPAN. b Schematic representation of RAD23B mutants. c Following lentiviral infection, IMR90 cells were incubated in HBSS for 6 hrs and harvested for immunostaining for Myc-RAD23B (anti-Myc) and PSMD7. Right, Estimation of the number of cells with SIPAN is indicated (representative from 4 independent experiments). d–f RAD23B undergoes LLPS in vitro. e Purified RAD23B in 50 mM HEPES pH 7.2 and 100 mM NaCl was mixed with Ficoll 400 and phase separation was visually observed, as RAD23B solution become turbid immediately after solution mixing (representative from 3 independent experiments). f RAD23B droplets were observed by bright-field microscopy. A portion of RAD23B and Ficoll 400 mixture was added on a microscope slide and a coverslip was applied on the solution near the edge of another coverslip to create space and allow liquid movement (representative from 3 independent experiments). g Effects of Ficoll and protein concentration on His-RAD23B droplet formation. Different concentrations of His-RAD23B were mixed with various concentration of Ficoll (representative from 3 independent experiments). h RAD23B was mixed with PEG 6000 or Dextran 60,000–90,000 and was added on a microscope slide and covered with a coverslip. Crystal violet was used to stain the droplets (representative from 3 independent experiments). i Inhibition of weak hydrophobic interactions alters RAD23B droplet formation. His-RAD23B was mixed with different concentrations of 1,6-hexanediol and analyzed for droplet formation (representative from 3 independent experiments). j RAD23B droplet fusion events during phase separation in vitro. Fusion events were detected by microscopy (representative from 3 independent experiments). k Coomassie showing purification of RAD23B protein and its corresponding mutants. l His-RAD23B mutants lacking critical domains were analyzed for droplet formation. Fewer droplets are observed with UBA1/2 mutant of His-RAD23B (representative from 3 independent experiments). Data in c represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant; 2-sided unpaired Student’s t-test is used. Source data are provided as a Source data file.