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. 2019 Sep 25;15(9):e1008380. doi: 10.1371/journal.pgen.1008380

Fig 6. Zw10 interacts with CENP-A loading factor CAL1.

Fig 6

A. Yeast two-hybrid interaction tests using SAC proteins as prey with either CAL1 or CENP-A as bait. The blue color indicates an interaction between prey and bait. B. Left panel. Coomassie showing purified GST-Zw10 and His-CAL1. Right panel. Immunoblot showing GST-pulldown assays of His-CAL1. C. Co-immunoprecipitation of CAL1 with GFP-Zw10 using the GFP-binding protein (GBP). Pulled down fractions were analyzed for the presence of CAL1. D. Time-lapse imaging of H2B-GFP/mCherry-tubulin expressing cells after 72 h Zw10 depletion. Imaging: 16 h. Time-lapse: 3 min. Scale bar: 2 μm. E. Quantification of mitosis duration shown in D. Mean +/- SEM, n>200 cells. Student’s t-test (***: p<0.001). F. SNAP-CENP-A incorporation in Zw10-depleted cells. After 72 h dsRNA treatment, a Quench-Chase-Pulse experiment (scheme in Fig 1H) was performed to stain newly synthesized SNAP-CENP-A molecules (red). DNA (DAPI) is shown in grey. Scale bar: 2 μm. G. Quantification of F showing the total SNAP-CENP-A centromeric intensity per nucleus as % of control. Mean +/- SEM of 3 experiments (n>300 cells), Student’s t-test (***: p<0.001).