Mechanism of homology search expansion during recombinational DNA repair. Dumont et al.

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Raw western blot images showing depletion of Pol3-AID and Rfc1-AID upon auxin treatment, in Figure S8D

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Cells were arrested in G1 with alpha-factor for 3 hours, and released in S-phase in YEP-Lactate-galactose media. Transcriptional inhibition was induced upon 50 ug/mL doxycycline and protein depletion induced upon 1.5 mM IAA addition 1 hour after S-phase release and HO induction, to allow for bulk DNA replication to take place. Protein extracts for western blot were prepared from 5.107 to 108 cells. Cells were lysed in cold NaOH buffer (1.85 N NaOH, 7.5% v/v beta-Mercaptoethanol) for 10 min in ice. Addition of trichloroacetic acid (15% final) for 10 min in ice allowed protein precipitation. After centrifugation at 15,000 g for 5 min, the pellets were resuspended in 100 µL of SB++ buffer (180 mM Tris-HCl pH 6.8, 6.7 M Urea, 4.2% SDS, 80 µM EDTA, 1.5% v/v Beta-mercaptoethanol, 12.5 µM Bromophenol blue). Denaturation was performed by heating 5 min at 65°C. Pre-cleared extracts were resolved on 12% precast polyacrylamide gel (Bio-Rad, cat. 4561043) and blotted on a PVDF membrane (GE Healthcare, cat. 10600023). Membranes were probed with mouse anti-AID antibody diluted at 1:1000 (CliniSciences, M214-3), or anti-GAPDH antibody diluted at 1:10000 (Invitrogen, MA515738), and revealed with an HRP-conjugated anti-mouse IgG antibody diluted at 1:10000 (Abcam, ab6789) using Immobilon Forte western HRP substrate (Merck, WBLUF0100) and a Chemidoc MP Imaging system (BioRad).

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