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. 2023 Jul 5;13:1204707. doi: 10.3389/fcimb.2023.1204707

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Copyright © 2023 Bachmaier, Gould, Polatoglou, Omelianczyk, Brennand, Aloraini, Munday, Horn, Boshart and de Koning

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Cyclic AMP affinity purification of CARPs with predicted cAMP binding domains. (A) CARP1 was pulled down by 8-AHA-cAMP agarose from T. brucei cells overexpressing CARP1 (2 replicates). (B) CARP11 was expressed in E. coli with an N-terminal His₆-SUMO tag and pulled down by 2-AHA- (top) or 8-AHA-cAMP agarose (bottom). The Western blots show the input fractions and the pulled down material in presence of increasing concentrations of cAMP (0 nM – 50 µM) for competition. The load of the bound fractions relative to the input is 15× for CARP1 and 12.5× for CARP11. Binding curves from the fluorescence quantifications are shown below; the amount of CARP pulled down without competition was set to 100%.