Fig. 2: Q586B2 is expressed in intracellular vesicles throughout different infection stages and in various trypanosomatid species.

a Immunofluorescence microscopy showing representative images of purified, fixed, and permeabilized T. b. brucei (AnTat1.1E) parasites stained with dapi (i.e., nucleus) alone or in presence of anti-Q586B2 Nbs (Nb27 and Nb39) or an anti-VSG Nb (Nb33). b Co-localization experiments using fixed V5-tagged T. b. brucei (90:13 strain) parasites stained with different primary antibodies (1:100 \({{{{{\rm{\alpha }}}}}}\)-V5 IgG, 1:1000 \({{{{{\rm{\alpha }}}}}}\)-p67 IgG, 1:1000 \({{{{{\rm{\alpha }}}}}}\)-BiP IgG and 1:50 Nb39), and after washing with fluorochrome-conjugated secondary antibodies. After washing, samples were mounted with ProLong Gold Antifade with DAPI mounting medium. The images were taken using a Zeiss Axio Imager M2 wide field (scale bar: 2.5 µm). Results of 2 independent experiments performed in triplicate are shown in (a) and (b). c Representative flow cytometric anti-Q586B2 Nb binding profile using Nb39 on different purified, fixed, and permeabilized parasites (Gating and conditions as described in Fig. S4b). (i) Parasites used are T. b. brucei bloodstream forms (AnTat1.1E), procyclic forms and metacyclic forms. Signals of parasites without staining (red), parasites in presence of Alexa-488 labeled anti-HA IgG alone (blue), and parasites in presence of Nb39 and Alexa-488 labeled anti-HA IgG (orange) are shown. (ii) Parasites used are T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. evansi, T. congolense, T. vivax, T. cruzi, L. major, L. infantum and Plasmodium falciparum. Signals of parasites in presence of Alexa-488 labeled anti-HA IgG alone was used as negative control (black/gray). c (right panel) Representative dot plot of 3 independent experiments showing the median fluorescence intensity (MFI) of anti-Q586B2 Nb39 following binding to fixed and permeabilized parasites. Each condition consists of technical duplicates (n = 2).