(A) The depletion assay was used to assess the salt-sensitivity of PEG-induced phase separation side-by-side with DNA-induced phase separation. ‘LOAD’ is a load control, ‘-’ is in the absence of DNA and PEG, ‘D’ indicates the addition of DNA, and ‘P’ indicates the addition of PEG-3350. Cdt1 phase separation was assessed at 75, 150, and 300 mM KGlutamate (‘KGlut’). (B) Comparison of initiator IDR hydropathy (dashed line indicates the average hydropathy of the Orc1, Cdc6, and Cdt1 IDRs) to the hydropathy of predicted ordered domains (‘Order’) and disordered domains (‘Disorder’) proteome wide. (C) Percent difference in initiator IDR amino acid composition relative to all predicted ordered (top) and disordered (bottom) domains proteome wide. The amino acids are ordered from high to low hydropathy (left to right) and those with a positive hydropathy value are indicated by a green background. (D) Comparison of initiator IDR fraction Leu, Ile, and Val (dashed line indicates the average fraction-L,I,V of the Orc1, Cdc6, and Cdt1 IDRs) to the fraction-L,I,V of all predicted ordered domains (‘Order’) and disordered domains (‘Disorder’) proteome wide. (E) To assess the distribution of branched hydrophobic residues, the fraction-L,I,V was calculated over a 5-residue sliding window for the Cdt1 IDR (top). Two mutants were made with either half (Cdt1Ile/Leu(0.5)) or all (Cdt1Ile/Leu(1.0)) of Leu and Ile residues mutated to Ala, and the distribution of L,I,V was calculated for each (middle and bottom, respectively). (F) The depletion assay was used to assess the phase separation capacity of Cdt1Ile/Leu(0.5) (top gel) and Cdt1Ile/Leu(1.0) (bottom gel) in the presence of increasing concentrations of PEG-3350 (0–12%, wt/vol). The fraction of protein retained at each concentration of PEG-3350 was quantitated. A t-test was used to calculate whether the different PEG concentrations resulted in a significant change in pelleting relative to the no PEG control. No significant (‘NS’) dose-dependent trend was identified for the effect of PEG on Cdt1Ile/Leu(0.5) (grey bars) or Cdt1Ile/Leu(1.0) (white bars) phase separation. Gel images are representative from three independent experiments.