Volume 29, 2022
|Number of page(s)||14|
|Published online||09 March 2022|
TbKINX1B can hydrolyse ATP in vitro. A. ATPase activity using the ELIPA in vitro assay. Kinesin activity was measured by the amount of generated phosphate per minute, in the presence of taxol-stabilized microtubules and ATP. The human kinesin heavy chain motor domain (KHCMD) is the positive control kinesin in presence of MTs (+MTs, black line) or negative control in the absence of MT (−MTs, grey line), the TbKINX1BMD (green), TbKINX1BMD deleted for the ATPase domain (ΔP-loopMD, red line), and 6HisTRX purified protein (yellow line), (n = 3). B. Recombinant TbKINX1B proteins purification. Western blot analysis (upper panel) and Coomassie-stained SDS-PAGE (lower panel) of the purification of the Trx-6HisTbKINX1BMD (A), Trx-6HisTbKINX1B-ΔP-loopMD (B), and Trx-6His (C) proteins. The recombinant proteins were immunolabelled using anti-Histidine tag. Abbreviations: Total (T), non-induced (NI), induced (IND), supernatant (S), flow-through (FT), wash (W, different wash fractions numbered), Elution (E, fractions numbered).
Impact of RNAi knockdown of TbKINX1B or TbBILBO1 in PCF on expression and localization. A. Minor phenotypes were observed in TbKINX1BRNAi cells induced for 24 h to 72 h. (a) The flagella were labelled with anti-PFR antibody (L8C4). (b) The FPC structures were labelled with anti-TbBILBO1 antibody. (c) The axonemes were labelled with anti-TbSAXO antibody (mAb25). (d) The FAZ structure was labelled with the anti-FAZ antibody (L3B2). Scale bars 5 μm. B. Expression level of TbKINX1B upon RNAi down-regulation of TbBILBO1. Immunoblotting of TbKINX1B, TbBILBO1 and Tubulin in whole cell protein extracts (WC), detergent-extracted cytoskeleton fraction (CSK), and soluble fraction (S) of lysed TbBILBO1RNAi non-induced (NInd) or induced for 36H (Ind) cells.
© D. Perdomo et al., published by EDP Sciences, 2022
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