Fusion fx7

We used radioimmunoprecipitation assay (RIPA) buffer containing a proteinase and phosphatase inhibitor cocktail (Cell Signaling, #5872, Danvers, MA, USA) for cell lysis for 20 min, followed by DNA shearing by sonication. The following antibodies were used for Western blot experiments: CUL4A, Cell Signaling, Danvers, MA, USA, CUL4B, Sigma HPA011880, Buchs, Switzerland, actin, Santa Cruz I19, Heidleberg, Germany, tubulin, Abcam ab6046 Cambridge, UK, CDT1, Cell Signaling, Danvers, MA, USA, p21 BD Bioscience, San Jose, CA, USA, cleaved caspase-3, Cell Signaling, Danvers, MA, USA, and phospho-histone H3 (Ser10) (pH3), Cell signaling, Danvers, MA, USA. Western blot imaging and signal quantification was performed using Fusion FX7 (Witec AG, Sursee, Switzerland). Uncropped blots with molecular weight markers are provided as Figure S7.

The in vitro SUMOylation assay was conducted using a commercial kit from Abcam (ab139470) using 1 μM target protein. The mixtures (E1, E2 ubc9, SUMO1, ATP-Mg, and target proteins) were set up in 20 μl of 10× SUMO reaction buffer and incubated at 37°C for 2 h. The reactions were collected every 20 min to study the progression of the SUMOylation process. The reaction mixtures were subsequently separated using SDS–PAGE (4–12%) and were subjected to Western blot analysis using anti SUMO1 antibody. Gels were scanned with a Fusion FX 7 (Witec) and analyzed using ImageJ (74 (link)). Experiments were performed in triplicate (un-cropped gels are shown in Fig S7).

Organs (lung, spleen, thymus) were lysed using DISC lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 1% (v/v) NP-40, 2 mM EDTA, 5 mM EGTA, 30mM NaF, 40 mM b-glycerophosphate pH 7.2, 10 mM sodium pyrophosphate, 2 mM activated sodium orthovanadate, protease inhibitors). The insoluble fraction of the lysate was pelleted by centrifugation and removed. Lysates were boiled and run on 4–20% Bis-Tris Gel NuPAGE using MOPS buffer (ThermoFisher Scientific). Proteins were then transferred onto PVDF-membrane (0.2 µm, ThermoFisher Scientific) using the Trans-Blot^® Turbo™ Transfer System (Bio Rad), according to the manufacturer’s instruction. After blocking with PBST containing 5% (w/v) skim milk, membranes were incubated with the indicated primary antibody in PBST containing 5% skim milk overnight at 4 °C, followed by incubation with the respective secondary antibody. Protein level expression was visualized on a film (Kodak, Rochester, NY, USA) using chemiluminescence (WesternBright ECL, Advansta, San Jose, CA, USA) or FusionFX7 (Witec AG, Heitersheim, Germany) with an aperture of 0.84 and binning 1 × 1.