Extended Data Fig. 4 | Changes in ubiquitylation levels and intranuclear
structure in response to hyperosmotic stress. a, HCT116 cells treated with
0.2 M sucrose for the indicated times were subjected to immunoblotting with
indicated antibodies. Representative results from two (anti-K48) or three
(anti-Ubiquitin) independent experiments. b, Mass spectrometry screening of
ubiquitylated substrates by the double-concentration method using TR-TUBE
and anti-diGly antibody. PSMB2-eGFPKI/KI cells were stimulated with 0.2 M
sucrose at the indicated times. The numbers of peptide spectrum matches for
each identified protein are shown as a heat map. Similar results were obtained
from two independent experiments. See Source Data for identified peptide
list. c, Correlative light and electron microscopy (CLEM) analysis of PSMB2-
FusionRedKI/KIeGFP-UbA AV S1 /AV S1 cells. Scale bars, 10 μm. Right, square enlarged
images on the left. The places where proteasome foci exist are circled in white.
Scale bars, 0.5 μm. Representative images from two independent experiments.
d, Time course images of PSMB2-eGFPKI/KI cells stimulated with 0.2 M sucrose,
with or without pre-treatment with 1 μM MLN-7243 or 50 μM MG-132 for 1 h.
Scale bars, 1 μm. Two independent experiments were performed with 0 min,
30 min, 240 min and MG-132, with similar results. e, Schematic of northern blot
probes and positions of the pre-rRNA processing intermediates discussed in
this Article. RNA was extracted from HCT116 cells stimulated with 0.2 M
sucrose for the indicated times and analysed by northern hybridization using
probes specific for the 5′ end and the middle of ITS1 and ITS2. Representative
results from three independent experiments. Gel source data for a and e are
shown in Supplementary Fig. 1.