Protease inhibitors

The analysis of DNA binding by Cbf11 was described in detail previously [22 (link)]. Briefly, cells were harvested at the density of 2.5 × 10⁷ cells/ml by centrifugation, washed with STOP buffer (150 mM NaCl, 50 mM NaF, 25 mM HEPES, 1 mM NaN₃; pH 8) and kept at -80°C. Native extracts were prepared in Lysis/Gelshift Buffer (25 mM HEPES, 0.1 mM EDTA, 150 mM KCl, 0.1% Triton X100, 25% glycerol, 1M urea, 2 mM DTT, FY protease inhibitors [Serva]; pH 7.6) by breaking the cells with glass beads in a FastPrep24 instrument (MP Biomedicals). Binding to radiolabelled double-stranded DNA probes containing CSL binding sites was detected as slow-migrating bands on a large native 5% polyacrylamide TBE gel. Signal intensities were quantified using the ImageQuant TL 7.0 software (GE Healthcare).

Cells were washed in PBS and harvested in radioimmunoprecipitation assay buffer (Table S3) enriched with protease inhibitors (Serva, Heidelberg, Germany) and phosphatase inhibitor cocktail (MilliporeSigma). The protein concentration was determined using a DC (detergent‐compatible) protein assay (Bio‐Rad, Hercules, CA, USA). The cell lysates were diluted to the same concentrations and mixed with loading buffer (150 mmol·L⁻¹ Tris–HCl pH 6.8, 3% SDS, 0.03% bromophenol blue, 30% glycerol, 3% β‐mercaptoethanol). Equivalent protein quantities were separated by SDS/PAGE and transferred onto polyvinylidene difluoride membranes (MilliporeSigma). The membranes were blocked in Tris‐buffered saline (TBS) containing 0.1% Tween‐20 and 5% nonfat dry milk for 1 h. The membranes were washed with TBS–Tween and incubated with specific primary antibodies overnight at 4 °C. The following primary antibodies were used: CHK1, pCHK1 (S296), pCHK1 (S345), phosphorylated γH2A.X (pH2AX; S139), and β‐actin. The membranes were washed and then incubated with secondary anti‐mouse IgG or anti‐rabbit IgG (GE Healthcare, Chicago, IL, USA) antibodies for 1 h. Detection of antibody reactivity was performed using chemiluminescence substrate Immobilon Western HRP Substrate (MilliporeSigma) and ChemiDoc™ Imaging System (Bio‐Rad). Dilutions, catalog numbers, and producers are listed in Table S3.

Sequences coding for H6-BubR1, H6-Bub1, and untagged Bub3 constructs were sub-cloned into pFLMultiBac vectors and baculoviruses were generated. Baculovirus expressing Bub3-TRX was generated by the Dortmund Protein Facility (DPF) using the pOPIN vector system {Berrow:2007cy}. Bub1/Bub3 and BubR1/Bub3 complexes were generated by co-infection and co-expression at 27°C for 72 hr. Insect cells were harvested by centrifugation at 1500 rpm for 30 min in a Sorvall RC 3BP+ (Thermo Scientific) centrifuge with Rotor H6000A, the pellets were frozen in liquid nitrogen and stored at −80°C. 1 g of cell pellet was re-suspended in 10 ml Lysis buffer (50 mM HEPES-KOH pH 7.5, 150 mM KCl, 15 mM imidazole, 2 mM DTE, 0.05% Tween20, PMSF, protease inhibitors [Serva]). Cells were lysed by sonication and centrifuged at 100000×g for 1 hr at 4°C. The supernatant was filtered through Nalgene bottle-top filter. The complexes were isolated from the cleared lysate on a 5-ml HisTrap (GE Healthcare) column. Peak fractions were pooled concentrated using Amicon concentrators and further purified in GF buffer (50 mM Hepes-KOH pH 7.5, 150 mM KCl, 2 mM DTE, 0.05% Tween20) by size exclusion chromatography using S200 16/60 column (GE Healthcare). Peak fractions were pooled, concentrated to typically 3 to 5 mg/ml, and frozen in liquid nitrogen.