Chemidoc mp scanner

Cell protein was harvested using RIPA buffer (G Biosciences, St. Louis, MO, USA) supplemented with Halt protease inhibitor (Thermo Fisher Scientific, Grand Island, NY, USA). Protein concentration was measured using the DC protein assay (Bio‐Rad, Hercules, CA, USA). Electrophoretic separation of protein (12–20 μg/well) was performed using 4–15% gradient polyacrylamide gels (Bio‐Rad). Separated protein was transferred onto PVDF membranes (Bio‐Rad), which were then blocked for 1 h at room temperature in Tris‐buffered saline containing 0.1% tween (TBS‐T) with 5% fat‐free milk (Bio‐Rad), followed by overnight incubation at 4°C with rabbit anti‐human PD‐L1 antibody (Cell Signaling Technologies, #13684T, Danvers, MA, USA) (1:1000 dilution) or mouse anti‐human β‐actin antibody (1:10 000 dilution) (Cell Signaling Technology, #3700 S) in 5% fat‐free milk with TBS‐T. Membranes were washed in TBS‐T and incubated for 20 min at room temperature with a 1:2000 dilution of horseradish peroxidase‐conjugated goat anti‐rabbit antibody (Promega, #W4011, Madison, WI, USA) or rabbit anti‐mouse antibody (Cell Signaling Technology, #7076 S) in 5% milk with TBS‐T. Protein signals were developed using the WesternBright ECL HRP substrate (Advansta, #K‐12045, San Jose, CA, USA) and measured using a Chemi‐Doc MP scanner (Bio‐Rad).

For electrophoretic mobility shift assay, 1 µM fC7L.1 was separately added with 150 nM of polyA pre-crRNA (pA3) and non-specific RNA in the buffer containing 20 mM Tris pH 7.5, 70 mM NaCl, 5% glycerol, and 25 µg/mL heparin. The reaction was incubated for 30 min at 37°C. After the reaction was complete, the samples were mixed with ×6 loading dye (B7025S, New England Biolabs) and run on a 10% TBE gel (EC62752BOX, Thermo Fisher Scientific) at 4°C at 150 V. Gels were stained with SYBR Gold II (Invitrogen) and scanned by Bio-Rad ChemiDoc MP scanner.

The 80-nt Oligo1 (100 nM) was first coated with purified yeast or human RPA (150 nM) in the reaction buffer containing 50 mM Tris-HCl, pH 7.5, 2 mM MgCl₂, 2 mM ATP, 35 mM KCl, 1 mM dithiothreitol (DTT), and 100 µg/ml BSA in a final volume of 10 μl at 37 ˚C for 10 min. Following this, purified yeast or human Rad51(1.6 µM) together with Bre1 (RNF20N) or mutated Bre1-EK2A (0.8 or 1.6 µM) were then added to the reaction and incubated at 37 ˚C for 10 min. To test the effect of Srs2 or FBH1 on strand exchange, 0.4 µM of Srs2 or FBH1 was added to the reaction together with Rad51. The strand exchange reaction was initiated by adding 50 nM of a Cy3′-labeled 40-nt dsDNA. After incubation at 37 ˚C for 30 min, samples were deproteinized by the addition of 5 μl of 1% SDS solution containing proteinase K (1 mg/ml) at 37 ˚C for 10 min and analyzed by electrophoresis (10% polyacrylamide gel in 1xTBE buffer). Signals were imaged with a ChemiDOC MP scanner (Bio-RAD) and quantified with the Image J software. DNA substrates for DNA strand exchange:
Oligo1: 5′-TTATGTTCATTTTTTATATCCTTTACTTTATTTTCTCTGTTTATTCATTTACTTATTTTGTATTATCCTTATCTTATTTA-3′.Oligo2: Cy3-5′TAATACAAAATAAGTAAATGAATAAACAGAGAAAATAAAG-3′
Oligo3: 5′-CTTTATTTTCTCTGTTTATTCATTTACTTATTTTGTATTA-3′
The dsDNA substrate was obtained by annealing the oligo2 and oligo3. The Cy3′ labeled duplex substrate was purified from a 10% polyacrylamide gel.