Sapphire capture software

A gel shift DNA binding assay used two complementary 50-mer oligonucleotides labeled at the 5′-end with either Cy3 or Cy5. The indicated concentration (5 or 3.6 μM) of Redβ or LiRecT in PBS (or cryo-EM buffer defined below) was mixed with 25 μM (nt) of the indicated oligonucleotide and incubated at 37 °C for 15 min. For some samples as indicated on the gel (lanes labeled “35”, “ad” or “nc”), a second oligonucleotide was added and incubated for an additional 15 min at 37 °C. For all samples the total reaction volume was 30 μl. For visualization 17.5 μl of each complex was mixed with 7.5 μl Orange G dye (65% w/v sucrose, 10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.3% Orange G powder from Sigma Life Sciences), loaded onto a 0.8% agarose gel and electrophoresed in 1× TBE at room temperature for 72 min at 96 V. Gels were imaged using a Sapphire Biomolecular Imager (Azure Biosystems) with Sapphire Capture Software (version 1.12.0921.0). Scanning parameters for Fig. 8 were pixel size 100 μm, scan speed high, 2.38 mm focus, intensity 2 for Cy5, intensity 4 for Cy3, black lighting 50, white 37186, gamma 1.37. Scanning parameters for Supplementary Fig. 1a, b were intensity 1 for Cy5, intensity 2 for Cy3, black lighting 50, white 15362, gamma 0.88.

Cells were harvested in RIPA buffer supplemented with complete protease inhibitor (Roche). Protein was run on NuPAGE Bis–Tris precast polyacrylamide gels (Thermo Fisher Scientific) alongside PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) and transferred to Odyssey nitrocellulose membrane (LI-COR Biosciences). Membranes were blocked in Intercept (PBS) Blocking Buffer (LI-COR Biosciences) before overnight incubation at 4°C with primary antibodies diluted in Blocking Buffer containing 0.2% Tween 20. Membranes were incubated with IRDye-conjugated secondary antibodies (LI-COR Biosciences) for 1 h and fluorescent signal visualized using a Sapphire Biomolecular Imager (Azure Biosystems) and Sapphire Capture software (Azure Biosystems). When appropriate, membranes were stripped with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) before being re-probed.