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. 2016 May 11;12(5):e1005618. doi: 10.1371/journal.ppat.1005618

Fig 7. Translocation intermediates are in a complex with EXP2.

Fig 7

(A-B) Representative live fluorescence (A) or IFA (B) images of the cell line expressing endogenous EXP2 fused to 3xHA (EXP2-3xHAendo) and SBP1-mDHFR-GFP (episomal) grown with (+WR) and without WR (control). DIC, differential interference contrast. Size bars: 5 μm. (C) IP of DSP-treated EXP2-3xHAendo parasites (+) using HA binding beads compared to 3D7 parasites (-). Released crosslink eluates (ReCLIP) and NaOH eluates (Eluate2) were separated by SDS-PAGE and silver stained. Asterisk, shows EXP2-3HA; arrowheads show antibody chains. The table shows the 20 top hits after mass spectrometry analysis of a ReCLIP and an Eluate 2. Peptides/control show peptide counts of the indicated protein over 3D7 (ReCLIP and Eluate 2 peptide counts pooled). SP, signal peptide; TM transmembrane domain. (D) Western blots of a representative IP experiment using HA binding beads show co-purification of SBP1-mDHFR-GFP (asterisk) in parasites grown with WR but not in untreated controls. Input, total lysate before IP; post, lysate after IP. (E) Western blots of a representative IP experiment as in (D) but using GFP binding beads to bind SBP-mDHFR-GFP. Asterisk: enrichment of co-purifying EXP2-3xHA in parasites grown with (+WR) compared to control (no WR). (F) Quantification of the signal intensity of the IPed EXP2-3xHA and the co-purifying SBP1-mDHFR-GFP (left, asterisk comparing SBP1-mDHFR-GFP in WR+ over its control: p = 0,0288; paired, two-tailed t test; n = 3) or the IPed SBP1-mDHFR-GFP and the co-purifying EXP2-3xHA (right, asterisk comparing EXP2-3xHA in WR+ over its control: p = 0,035; paired, two-tailed t test. Asterisk comparing EXP2-3xHA enrichment with SBP1-mDHFR-GFP enrichment: p = 0,047; unpaired, two-tailed t test; n = 3) in parasites grown with (WR+) or without (control) WR. Error bars show S.D., n.s., not significant. (G) Western blots of a representative IP experiment with REX2-mDHFR-GFP expressed in EXP2-3xHAendo parasites. REX2-mDHFR-GFP is not enriched in parasites grown with WR over untreated controls. Input, total lysate before IP; post, lysate after IP.