Figure 8. CENP‐C cupin dimerisation is critical for CAL1 binding.

• A–C
  SEC analysis of (A) His‐CENP‐C_{1264–1411 F1324R} and His‐SUMO‐CAL1_841–979, (B) His‐CENP‐C_1264–1411 and His‐SUMO‐CAL1_{841–979 I900R/K907A/Y908A} and (C) His‐CENP‐C_{1264–1411 L1357E/M1407E} and His‐SUMO‐CAL1_841–979. Samples were analysed using a Superdex 75 in 20 mM Tris–HCl pH 8.0, 0.1 M NaCl and 2 mM DTT. Corresponding fractions shown by SDS–PAGE with Coomassie stain underneath.
• D
  Representative IF images and quantification of tethering assays. U2OS cells containing a LacO array were transfected with GFP‐LacI‐CENP‐C with CAL1‐V5 to assess interaction. CENP‐C mutants F1324R, L1357E/M1407E and CAL1 mutant I900R/K907A/Y908A were tested in each construct separately. Scale bar: 10 μm (n = 4 experiments except F1324 where n = 3 experiments).
• E
  Representative IF images and quantification of in vivo tethering assays. Drosophila Schneider S2 cells containing a LacO array were transfected with GFP‐LacI‐CENP‐C and CAL1‐HA to assess interaction. Arrows point to the LacO site. Scale bar is 5 μm (n = 3 experiment, except CAL1 1900R/K907A/Y908A where n = 2 experiments).

Data information: In (D), data presented as mean ± SEM of 3 or 4 experiments, n ≥ 22 cells per experiment. P‐values were calculated using an Mann–Whitney test. In (E), data presented as mean ± SEM of 3 experiments, except for CAL1 mutant I900R/K907A/Y908A which was 2 experiments. n ≥ 44 cells per experiment, P‐values were calculated using Mann–Whitney test (****P < 0.0001).