Cells were lysed in NP-40 Lysis Buffer (50 mM Tris-HCl, 150 mM
NaCl, 5 mM
EDTA, 2% NP-40, pH 8.0)
supplemented with protease inhibitor (complete ULTRA tablets EASYpack, Roche)
and phosphatase inhibitor (PhosSTOP, Roche). Protein yield was determined by
Bradford assay (Biorad). Total protein (30–50 μg)
was separated on NuPAGE SDS Gels (Life
Technologies) and tank-blotted to nitrocellulose membranes. Following blocking
in Tris Buffered Saline with Tween 20 (TBST; 5 mM Tris, 15 mM NaCl, 0.1% Tween 20, pH 7.5) with 10%
nonfat dry milk or 5% bovine serum albumin for 1 h, membranes were
incubated with primary antibodies diluted in TBST/5% nonfat dry milk or TBST/5%
bovine serum albumin and incubated overnight at
4 °C. Antibodies: α-p53 (SantaCruz
DO-1, 1:10,000); α-PLK1 (SantaCruz F-8, sc-17783, 1:200);
α-Mdm2
(Hybridoma supernatant (4B2), 1:2); α-p21 (SantaCruz C-19, sc-397, 1:200);
α-Phospho-Histone H3
(Ser10) (Cell Signalling #9701, 1:200);
α-GLuc
(Nanolight 401P, 1:1000); α-actin (Abcam AC-15,
1:5,000). Proteins were detected with secondary antibody (α-mouse
IgG-HRP, α-rabbit IgG-HRP from
GE Healthcare, 1:3,000) and ECL kit
(SuperSignal West Dura Chemiluminescent Substrate, Thermo Scientific). Uncropped
scans of representative Western blots are shown inSupplementary Fig. 9 .
NaCl, 5 mM
EDTA, 2% NP-40, pH 8.0)
supplemented with protease inhibitor (complete ULTRA tablets EASYpack, Roche)
and phosphatase inhibitor (PhosSTOP, Roche). Protein yield was determined by
Bradford assay (Biorad). Total protein (30–50 μg)
was separated on NuPAGE SDS Gels (Life
Technologies) and tank-blotted to nitrocellulose membranes. Following blocking
in Tris Buffered Saline with Tween 20 (TBST; 5 mM Tris, 15 mM NaCl, 0.1% Tween 20, pH 7.5) with 10%
nonfat dry milk or 5% bovine serum albumin for 1 h, membranes were
incubated with primary antibodies diluted in TBST/5% nonfat dry milk or TBST/5%
bovine serum albumin and incubated overnight at
4 °C. Antibodies: α-p53 (SantaCruz
DO-1, 1:10,000); α-PLK1 (SantaCruz F-8, sc-17783, 1:200);
α-Mdm2
(Hybridoma supernatant (4B2), 1:2); α-p21 (SantaCruz C-19, sc-397, 1:200);
α-Phospho-Histone H3
(Ser10) (Cell Signalling #9701, 1:200);
α-GLuc
(Nanolight 401P, 1:1000); α-actin (Abcam AC-15,
1:5,000). Proteins were detected with secondary antibody (α-mouse
IgG-HRP, α-rabbit IgG-HRP from
GE Healthcare, 1:3,000) and ECL kit
(SuperSignal West Dura Chemiluminescent Substrate, Thermo Scientific). Uncropped
scans of representative Western blots are shown in