Typhoon 5 imager

Reactions were pre-assembled into two separate mixtures with proteins diluted into reaction buffer containing 30 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl₂, 2 mM DTT and 2 mM ATP. CRL components and ³²P-labeled substrate protein were sequentially added (tube 1) and incubated for 5, 10, 20, 40 and 60 minutes. Meanwhile, E1, ubiquitin and UBE2R2 proteins were sequentially added and incubated for a period of approximately 15 minutes (tubes 2). Ubiquitylation reactions were initiated by mixing equal volumes of tube 2 mix with the tube 1 incubations and time points were collected at 10-second and 5-minute intervals. Reactions were quenched in reducing 2X SDS-PAGE loading buffer (100 mM Tris-HCl (pH 6.8), 20% glycerol, 30 mM EDTA, 4% SDS and 4% beta-mercaptoethanol). Each reaction was performed in triplicate and was resolved on 18% polyacrylamide SDS-PAGE gels. Autoradiography was performed using an Amersham Typhoon 5 imager and quantification performed with ImageQuant software (Cytiva). The fraction of substrate ubiquitylated was calculated as the fraction of products over the total signal of each respective lane.

Experiments were performed in 10 μl reactions by mixing together 30 nM 40N40-FAM nucleosome (601[canonical] or 601[TA-rich +1]) and 120 nM Chd1, in either nucleotide-free conditions or 1 mM AMP-PNP, in buffer containing 15 mM HEPES-KOH, pH 7.6, 50 mM KCl, 2.5 mM MgCl₂, 0.1 mg ml⁻¹ BSA, 1 mM DTT, and 5% sucrose. To each reaction, salmon sperm DNA (Thermo Fisher, cat #15632011) was added to a final concentration of 0, 0.0625, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 μg μl⁻¹. After a 15 min incubation at room temperature, 2 μl of each reaction was loaded on a 4.25% native PAGE gel. After electrophoresing for 90 min (0.25x TBE, 4°C, 100V), gels were scanned on a Typhoon 5 imager (Cytiva).

Nucleosome sliding reactions were carried out in the presence of remodeler enzyme with 40 nM FAM-80-601-0 or 0-601-80-FAM nucleosomes, using 2.5 mM ATP and 1x Slide buffer (20 mM HEPES-KOH, pH 7.6, 100 mM KCl, 5mM MgCl₂, 0.1 mg ml⁻¹ BSA, 1 mM DTT, 5% sucrose). Remodeler concentrations are as indicated in figure legends. After initiating each 30 μl reaction with addition of ATP, 1 μl aliquots were taken out at each time point and stopped by adding to separate tubes containing 7.5 μl quench buffer (20 mM Hepes-KOH, pH 7.6, 50 mM KCl, 0.1 mg ml⁻¹ BSA, 1 mM DTT, 5% sucrose, 40 mM EDTA, 2 μg μl⁻¹ salmon sperm DNA). For each experiment, 2.5 μl aliquots of each quenched reaction were resolved on 6% native PAGE, and scanned on a Typhoon 5 imager (Cytiva).