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. 2021 Jun 3;220(8):e202101022. doi: 10.1083/jcb.202101022

Figure S5.

Figure S5.

Analysis of KKT2 and KKT3 mutants in trypanosomes.(A) Full-length KKT2 proteins with indicated mutations were ectopically expressed with 10 ng/ml doxycycline (Dox) for 1 d. Cell lines: BAP327, BAP365, BAP366, BAP367, BAP368, and BAP369. Scale bar, 5 µm. (B) RNAi of KKT2 and KKT3 causes growth defects in T. brucei. Growth curves of KKT2 5′UTR RNAi (i), KKT2 5′UTR RNAi with YFP-KKT2 (resistant to the RNAi; ii), KKT3 3′UTR RNAi (iii), and KKT3 3′UTR RNAi with KKT3-YFP (resistant to the RNAi; iv). 1 µg/ml doxycycline was added to induce RNAi. Controls are uninduced cell cultures. Similar results were obtained from at least two independent experiments. Cell lines: BAP1554, BAP1681, BAP1555, and BAP1682. (C) Indicated KKT2 mutants colocalize with a kinetochore marker, tdTomato-KKT1. Cell lines: BAP2036, BAP2033, and BAP2035. (D) KKT2 CL domain mutants localize at kinetochores even when endogenous KKT2 protein is depleted (n > 50, 2K1N cells). One allele of KKT2 was mutated and tagged with an N-terminal YFP tag, and the other allele was depleted for 4 d using RNAi-mediated knockdown by targeting the 5′UTR of the KKT2 transcript. Maximum intensity projections are shown. RNAi was induced with 1 µg/ml doxycycline. Cell lines: BAP1681, BAP1789, BAP1779, and BAP1786. (E) KKT3C707A C710A colocalizes with tdTomato-KKT1, while KKT3C668A C671A and KKT3D692A mutants do not. Cell lines: BAP2037, BAP2038, and BAP2034. Scale bars, 5 µm.