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. 2020 Apr 1;219(6):e201907082. doi: 10.1083/jcb.201907082

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© 2020 Jackman et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S1. — Cyclin B1 binds to MAD1 through the acidic face of a helix encoded by exon 4. Related to Fig. 1. (A) Colloidal blue stained SDS-PAGE gel of cyclin B1 immunoprecipitated from HeLa cells. Marked bands were excised and identified by mass spectrometry. (B) Silver-stained SDS-PAGE gel of cyclin B1 immunoprecipitates from RPE cyclin B1-Venus^+/−: Ruby-MAD2^+/− cells (lane 1) and MAD1 E53K/E56K clone 7D2 (lane 2). (C) Cyclin B1 immunoprecipitates from HeLa cells expressing Flag-epitope tagged MAD1α or MAD1β or MAD1 with a mutated nuclear localisation sequence KKR79-82AAA (Mad1-NLS), probed with anti-FLAG (upper panel) or anti-cyclin B1 (lower panel) antibodies. (D) Heptad registration of acidic residues of MAD1 within coiled-coil configuration, predicted using PairCoil2. (E) Confocal image of HeLa cyclin B1-Venus^+/− (green) cells transfected with either MAD1 L51G/E52A/E53A/R54A/E56A-Ruby (left panel, red) or MAD1 E53/56K-Ruby (right panel, red). Scale bar, 10 µm.