Fig. 4.
Efficient assembly of cohesinADP/AlFx–DNA complexes requires binding of ADP-AlFx in both active sites. (A) DNA-bead assay to assess Walker A cohesin mutant binding. (Upper) Cohesin bound to DNA beads incubated with EDTA, ATP, or ADP/AlFx and detected by Western blot using anti-V5 antibodies (Psm3-3V5). (Lower) Quantitation of Western blots. (B) Protein-bead assay to assess Walker A cohesin mutant binding. (Upper) DNA bound to cohesin incubated with EDTA, ATP, or ADP/AlFx, after cohesin immunoprecipitation, and detected using ethidium bromide-stained agarose gel. (Lower) Quantitation of DNA.