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. 2019 Jul 1;30(14):1676–1690. doi: 10.1091/mbc.E19-03-0152

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© 2019 Chiurillo et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 6: — Functional mutagenesis of critical amino acid residues at the selectivity filter of TcMCU and TcMCUb. (A) Western blot analysis of total protein extracts of control (Scr) cells, TcMCU-KO (MCU KO), TcMCU-KO plus EV (KO + EV), TcMCU-KO plus TcMCU (KO + MCU), TcMCU-KO plus TcMCU^D221N (D221N), TcMCU-KO plus TcMCU^D223N (D223N), and TcMCU-KO plus TcMCU^E226Q (E226Q) epimastigotes using anti-c-Myc antibodies. Anti-tubulin antibodies (Tub) were used as a loading control. (B) Ca²⁺ uptake reconstitution in DIG-permeabilized epimastigotes. Experimental conditions were as in Figure 1D. (C) Quantification of data from three experiments as shown in B. Relative Ca²⁺ uptake at 500 s compared with controls (Scr). (D) Western blot analysis of total protein extracts of control and knock-in mutated cells, using anti-c-Myc antibodies. Anti-tubulin (Tub) antibodies were used as a loading control. (E) Ca²⁺ uptake in DIG-permeabilized control (Scr), TcMCUb-KO (MCUb-KO), and knock-in TcMCUb^control (MCUb WT), TcMCUb^D161N (MCUb D161N) and TcMCUb^E164Q (MCUb E164Q) epimastigotes. Experimental conditions were as in Figure 1D. (F) Quantification of data from three experiments as shown in E. Relative Ca²⁺ uptake at 500 s compared with controls (Scr). Values in C and F are means ± SD (n = 3); ***P < 0.001; one-way ANOVA.