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. 2021 May 11;22(7):e52295. doi: 10.15252/embr.202052295

Figure 2. hSgo1 binding to the LxxIxE binding pocket of PP2A‐B56 is required for cohesion protection.

Figure 2

  1. Structure of the reported PP2Aγ‐B56‐hSgo1 binding interface (top) and residues mutated in the B56α 5A mutant are shown (bottom).
  2. IP of YFP‐B56α from cells stably expressing the B56α WT, R222E, and 5A followed by immunoblotting of indicated proteins. Representative blots are shown (top). hSgo1 signals were normalized to YFP and plotted (bottom). Error bars represent SD (n = 4).
  3. Reciprocal IP of (B). YFP‐hSgo1 expression construct was transfected into cells stably expressing FLAG‐B56α WT, R222E and 5A, followed by YFP IP and immunoblotting of indicated proteins. Representative blots are shown (top). B56α signals were normalized to YFP and plotted (bottom). Error bars represent SD (n = 4).
  4. Representative images of chromosome spreads from the indicated conditions. Scale bar, 5 µm.
  5. Quantification of (D). The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 4 independent experiments and the mean and SD are indicated.
  6. hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere‐targeting domain (CB). Representative images of chromosome spreads are shown. CB targets all the rescue constructs (green) to the centromere. Scale bar, 5 µm.
  7. Quantification of (F). The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 3 independent experiments and the mean and SD are indicated.

Source data are available online for this figure.