(Tomy Seiko), and then collected by centrifugation (20,000g for 15 min
at 4 °C). The protein concentration was determined using the BCA pro-
tein assay kit (Thermo Fisher Scientific). For anti-Flag immunoprecipi-
tation, anti-DDDDK-tag mAb-Magnetic beads (Medical and Biological
Laboratories), M185-11) were used to precipitate Flag-tagged protein
complex from 1 mg of cell lysate by incubating for 1 h at 4 °C. After five
washes with buffer A containing 0.2% NP-40, the proteins were eluted
for 10 min at 70 °C in 1× NuPAGE LDS sample buffer.
SDS–PAGE and immunoblotting
Cells were lysed with buffer A with complete protease inhibitor cocktail
(EDTA free) (Roche) for 30 min on ice, and then centrifuged at 12,000g
for 5 min at 4 °C. Supernatants were collected, and protein concentra-
tion was measured using the BCA protein assay kit (Thermo Fisher
Scientific). Cell lysates were boiled in 1× LDS NuPAGE sample buffer
for 10 min, and then electrophoresed on 4–12% NuPAGE Bis-Tris gels
(Thermo Fisher Scientific). Proteins were transferred to polyvinylidene
difluoride membranes (Millipore). The membranes were blocked for
30 min in 5% non-fat milk, and then incubated 1 h at room temperature
with primary antibodies. The primary antibodies used were as follows:
anti-Flag mouse monoclonal (A8592; Sigma-Aldrich); anti-multi-ubiq-
uitin rabbit polyclonal (Z0458; Dako); anti-PSMB2 rabbit polyclonal
(BML-PW8890; Enzo Life Sciences); anti-PSMD6 rabbit polyclonal
(BML-PW8225; Enzo Life Sciences); Lys48-Specific anti-ubiquitin rab-
bit monoclonal (05-1307; clone Apu2; Millipore); anti-RAD23B rabbit
monoclonal (13525; Cell Signaling Technology); anti-UBE3A rabbit
polyclonal (10344-1-AP; Protein Tech); anti-cleaved caspase-3 (Asp175)
rabbit polyclonal (9661; Cell Signaling Technology); and anti-β-actin
pAb-HRP-DirecT (PD030; MBL). After extensive washing with TBST,
the membranes were immunoblotted with secondary antibodies for
30 min at room temperature. The following secondary antibodies
were purchased from Jackson ImmunoResearch Laboratories: HRP-
conjugated goat anti-rabbit Ig and HRP-conjugated goat anti-mouse
Ig. After washing several times with TBST, blots were developed using
ECL Prime Western Blotting Detection Reagent (GE Healthcare) and
analysed on an ImageQuant LAS4000 (GE Healthcare).
Northern blotting
Total RNA was isolated with TRIzol reagent (Thermo Fisher Scientific).
Total RNA (4 μg per lane) was resolved on a 1.2% formaldehyde agarose
denaturing gel in 1× TT (30 mM Tricine and 30 mM triethanolamine)
buffer by electrophoresis for 120 min at 200 V, followed by capillary
transfer to Hybond-N+ membrane (GE Healthcare) with 20× SSC (3 M
NaCl and 300 mM trisodium citrate dihydrate) for 18 h. After transfer,
RNA was UV cross-linked to the membrane using a CL-1000 ultraviolet
crosslinker (UVP) at 120 mJ cm^2. Membranes were pre-hybridized with
DIG Easy Hyb Granules (Roche) dissolved in double-distilled water
(hybridization buffer), for 1 h in a hybridization oven at 50 °C. DIG-
labelled DNA probes were added to the buffer and incubated for 20 h.
Membranes were washed with non-stringent buffer (2× SSC and 0.1%
SDS) for 15 min at 50 °C, followed by stringent buffer (0.1× SSC and 0.1%
SDS) twice for 15 min each. The membrane was then washed with 1× MA
buffer (100 mM maleic acid and 150 mM NaCl, pH 7.0) for 10 min at room
temperature, and incubated with Blocking Reagent (Roche) for 30 min.
Anti-digoxigenin–AP, Fab fragments (Roche) was added to the blocking
reagent, and further incubated for 1 h. After that, the membranes were
washed three times for 10 min each with 1× MA buffer containing 0.3%
Tween-20, and then equilibrated in buffer-A (100 mM Tris-HCl and 100
mM NaCl, pH 9.5). To detect RNA, the membranes were incubated with
CDP-star (Roche) for 10 min, and then chemiluminescence was visual-
ized on a LAS-4000 mini (GE healthcare). DNA probes were labelled
with DIG at the 5′ end; probe sequences were as follows: 5′ETS; 5′-CGGA
GGCCCAACCTCTCCGACGACAGGTCGCCAGAGGACAGCGTGTCAGC-3′;
ITS1; 5′-CCTCGCCCTCCGGGCTCCGGGCTCCGTTAATGATC-3′; and ITS2;
5′-CTGCGAGGGAACCCCCAGCCGCGCA-3′.
Purification of recombinant proteins
Codon-optimized human RAD23B or RAD23A cDNA (Eurofins Genom-
ics) was subcloned into Escherichia coli expression vector pGEX6P1 (GE
Healthcare), in which the Cys sequence (TGC) was inserted upstream
of the BamHI site for Cy labelling. RAD23B mutants, the UBL domain
(Δ1–79), or two UBA domains (Δ180–238/Δ353–409), were generated by
inverted PCR. Human ubiquitin with a N-terminal Met–Cys was inserted
into vector pET26b (Novagen). GST–Cys–RAD23 proteins or Met–
Cys-ubiquitin were expressed in E. coli BL21 (DE3) cells by induction with
0.2 mM IPTG at 28 °C for 3 h. GST-tagged proteins were adsorbed onto
glutathione–Sepharose 4B (GE Healthcare) in buffer B (50 mM HEPES-
KOH, pH 7.5, 100 mM NaCl and 10% glycerol) containing 0.1% Triton
X-100. After washing with the same buffer, the beads were incubated
with PreScission protease (GE Healthcare) to cleave the GST-tag from
GST–Cys–RAD23 proteins, and the Cys–RAD23 proteins were recov-
ered. Cells expressing Met–Cys–Ubiquitin were suspended in 50 mM
HEPES-KOH, pH 7.5 and 100 mM NaCl and lysed by sonication. After
centrifugation at 20,000g for 30 min, the supernatant was recovered
and boiled at 80 °C for 5 min. The lysate was further clarified by cen-
trifugation (20,000g for 20 min) to obtain Met–Cys–Ubiquitin. Cy3 or
Cy5 labelling was performed using Cy Maleimide Mono reactive dye (GE
Healthcare). After Cy labelling, Cy5–ubiquitin and Cy3–RAD23 proteins
were separated from free dye on a PD-10 column (GE Healthcare). Unan-
chored K48-linked ubiquitin chains and K63-linked ubiquitin chains
were prepared using UBE2K/E2-25K and Ubc13/Mms2, respectively,
as previously reported^41. To obtain Cy5-labelled ubiquitin chains with
defined lengths, we further purified the ubiquitin chains by Superdex
75 10/300 GL size exclusion chromatography (GE Healthcare).In vitro phase-separation assay
Droplet formation of the purified protein was monitored by fluores-
cence and differential interference contrast (DIC) microscopy using
an Olympus Fluoview FV3000 (described above). Unless otherwise
noted, 20 μM Cy5–ubiquitin and 20 μM Cy3–RAD23 proteins were
incubated at room temperature in 50 mM Tris-HCl (pH 7.5), 3% PEG
(Sigma-Aldrich) and 200 mM NaCl. The mixture in a total solution
volume of 10 μl was placed in a glass-bottom dish (MatTek) or Senso-
plate glass-bottom 384-well plate (Greiner Bio-One) coated with 0.1%
PVA (Sigma-Aldrich).Phase diagram mapping
After mixing RAD23B and ubiquitin proteins as described above,
the mixture was placed on Sensoplate glass-bottom 384 well plates
(Greiner) coated with 0.1% PVA (Sigma-Aldrich) and incubated for
90 min at room temperature. The presence or absence of droplets was
scored by Cy3 and Cy5 fluorescence in each well at 100× magnification
using an Olympus Fluoview FV3000. For fluorescence observation, the
maximum projection of three z-stacks from the bottom of the plate
was processed, and the scale was set in the range from 0 to 4,095. Two
wells were prepared per experiment, and if fluorescence was recorded
at three or more areas in each well, it was regarded as a droplet. The
experiment was performed twice.Quantification of RAD23B and RAD23A in HCT116 cells
Cellular abundance of RAD23A and RAD23B were determined by quan-
titative western blotting using recombinant protein as a standard.
After washing twice with PBS, HCT116 cells were lysed by sonication
in 2% SDS, 20 mM HEPES-Na (pH 7.5) and 1 mM EDTA. Proteins were
subjected to western blotting with anti-RAD23A antibody (24555; Cell
Signaling Technology) or anti-RAD23B antibody (13525; Cell Sign-
aling Technology). Copy number per cell and concentration were
calculated, assuming that total protein per cell was approximately
200 pg and that cell volume was approximately 1 pl, as described
previously^26 ,^42.