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. 2022 Sep 16;13:5445. doi: 10.1038/s41467-022-33108-z

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Indirect immunofluorescence analysis of CARP3 and ACP1-Ty1 (left) or ACP6-Ty1 (right) in procyclic T. brucei 29-13. Top: CARP3 (green) + DNA (blue, DAPI); middle: Ty1 (red) + DNA; bottom: merge. Scale bars 5 µm. b Western blot (top, probed with anti-CARP3 and anti-PFR-A/C as loading control) and SoMo assay (bottom) of procyclic T. brucei AnTat 1.1E CARP3-PAmCherry/∆carp3 cells. Wild type (WT) and homozygous carp3 knock-out (KO) serve as controls. M: molecular weight marker. c PALM imaging and (d) single molecule localization shown via centroids of CARP3-PAmCherry (PAmCh, red) and ACP1-mNeonGreen (mNG, green) at a procyclic T. brucei AnTat 1.1E flagellar tip. Corresponding phase contrast shown as inset in (c). Colocalization analysis of CARP3-PAmCherry with ACP1-mNeonGreen (e, f). CARP3 (e, left) and ACP1 (e, right) localizations are color-coded according to their respective Coordinate Based Colocalization (CBC) values, where a higher value signifies a higher colocalization probability. CBC histograms (f) of CARP3-PAmCherry (red) and ACP1-mNeonGreen (green) single molecule distributions. CBC values were calculated in a maximum radius of 300 nm and radius intervals of 5 nm. n = 19 flagella. g PALM imaging of CARP3-PAmCherry (red) and PDEB1-mNeonGreen (green) in a procyclic T. brucei AnTat 1.1E flagellum. PALM imaging (h) and colocalization analysis (i, j) of CARP3-PAmCherry (red) and calpain 1.3-mNG (green) at a procyclic T. brucei AnTat 1.1E flagellar tip. CARP3 (i, left) and calpain 1.3 (i, right) localizations are color-coded according to their CBC values. Scale bars in (c), (g) and (h) 0.5 μm. Quantification in (j) shows CBC histograms of CARP3-PAmCherry (red) and calpain 1.3-mNG (green) single-molecule distributions. n = 15 flagella. k, l Indirect immunofluorescence analysis of CARP3 localization in procyclic T. brucei AnTat 1.1 upon constitutive overexpression of CARP3-Ty1, CARP3(154-489)-Ty1 or CARP3(1-160)-Ty1 or in situ add-back of CARP3∆3 in a carp3 KO background. k Anti-CARP3 red, anti-Ty1 green, DAPI (DNA) blue. (l) mAB25 (anti-TbSAXO) red, anti-CARP3 green. Scale bars 5 μm. m SoMo assay of cell lines as in (k, l) including WT and carp3 KO. Source data to (b) is provided as Source Data file.