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. 2019 Feb 27;8(3):bio039818. doi: 10.1242/bio.039818

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© 2019. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 1. — Identification of parasite proteins that interact with the host Golgi apparatus. (A) Protocol for identifying parasite proteins that bind host membrane fractions. CHO cells were lysed, and after differential centrifugation, total membranes were layered on a discontinuous sucrose gradient. The Golgi apparatus and endoplasmic reticulum (ER) were collected. In parallel, parasite cytosolic fractions containing parasite proteins were also collected. Fractions B and C from the sucrose gradient, containing the enriched Golgi and ER membranes, respectively, were incubated with parasite lysate, and then pelleted and analyzed by mass spectrometry. (B) Western blot analysis after organelle fractionation. Antibodies specific to calnexin (ER marker) and giantin (cis-Golgi marker) were used. The anti-golgin antibodies recognize multiple glycoprotein forms, whereas the anti-calnexin antibodies bind a single protein. (C) Venn diagram summarizing mass spectrometry data from quadruplicate experiments, representing parasite proteins identified in the input parasite lysate (RH), host Golgi-enriched membranes, and host ER-enriched membranes. (D) Classification of the number of secreted proteins (rhoptry, dense granule and microneme) identified in the mass spectrometry data in the input parasite lysate, Golgi-enriched membranes, and ER-enriched membranes. Results are presented as numbers of proteins±s.d. (E) Identification of parasite proteins binding to host Golgi membranes. Immunoblots were probed with anti-HA (for TgROP13), anti-M2AP, and anti-GRA3 antibodies.