Sc 13125

For rescue experiments, cells were transfected with 100 ng HA-Ub^KEKS plasmid construct using JetPrime (Polyplus-transfection SA, France) according to the manufacturer's instructions. 24 h after transfection media was changed to fresh culture media and cells were further grown for 48 h. Cells were then prepared following the general protocol describe above. Primary antibodies against nucleolin (Abcam #ab136649, 1:2000), UBF (monoclonal F-9, Santa Cruz Biotechnology #sc-13125, 1:300) and fibrillarin (monoclonal 38F3, Abcam #ab4566, 1:1000) were used. Anti-mouse (Invitrogen #A11003, 1:800 was used as secondary antibodies.

The following primary antibodies were used. Mouse monoclonal antibodies anti-histone H2AX phospho-Ser139 (1/1000 Western, 1/200 immunostaining; Millipore-Upstate 05–636, MA, USA), anti-BrdU (1/25 immunostaining; Sigma B8434, UK), anti-WRAP53 (1/200 immunostaining; Abnova H00055135-M04, USA), anti-UBF (1/100 immunostaining; Santa Cruz Biotechnology SC-13125) and anti-B23 (1/100 immunostaining; Abcam ab10530). Rabbit polyclonal antibodies anti-histone H2AX phospho-Ser139 (1/200 immunostaining; Novus Biologicals NB100–384), anti-trimethyl-histone H4 (Lys 20) (1/250 immunostaining; Millipore-Upstate 07–463, USA), anti-53BP1 (1/100 immunostaining; Bethyl Laboratories A300-272A, Inc., USA), anti-CTCF (1/100 immunostaining; Millipore-Upstate 07–729, MA, USA), anti- Iba1 (1/500 immunostaining; Wako 019–19,741), anti-GFAP (1/500 immunostaining; Thermo Fisher Scientific PA3–16727), anti-NeuN (1/100 immunostaining; Abcam ab177487) and anti-Histone H3 (1/2000 Western; Thermo Fisher Scientific PA5–16183). Chicken polyclonal anti-β-galactosidase (1/200 immunostaining; Abcam ab9361). Specific secondary antibody conjugated with FITC or TexasRed were used (Jackson Lab., USA).