Figure EV2. Interaction between Cse4 and Okp1 (related to Figs 3D and 4).

1. Co‐immunoprecipitation of Okp1 and Cse4. Co‐IP was carried out in cells carrying CSE4 with (+) or without (−) 3xHA‐tag and OKP1 with (+) or without (−) 9xmyc‐tag. Cells were grown at 30°C, and Cse4 was immunoprecipitated using an α‐HA antibody (AEY2781, AEY5972, AEY6578). The presence of Cse4 and Okp1 in the immunoprecipitate was tested by Western blotting with α‐HA and α‐myc antibody, respectively. Left: inputs of the indicated strains; right: α‐HA immunoprecipitates of the indicated strains.
2. Two‐hybrid analysis of the interaction between Cse4 (aa 11–139) and Okp1. Cells carrying the indicated constructs were streaked on minimal medium (+His) or on medium lacking histidine (−His) to test for activation of the two‐hybrid reporter.
3. Activity of the lacZ two‐hybrid reporter of strains carrying the indicated two‐hybrid constructs.
4. Absence of in vitro interaction between Ctf19/Mcm21 and Cse4N. Recombinant Ctf19/Mcm21 and Cse4N were co‐incubated prior to size‐exclusion chromatography (SEC) as for Okp1/Ame1 in Fig 4A. Coomassie Blue‐stained SDS–PAGE gels of SEC runs with Mcm21/Ctf19 (top), Cse4N (middle) and co‐incubation of Ctf19/Mcm21 with Cse4N (bottom) are shown.

Source data are available online for this figure.