Rat α ha 3f10

Samples were resolved on 8% Tris/glycine polyacrylamide gels and transferred to a PVDF membrane (Millipore Immobilon, Merck, pore size 0.45 µm). The membranes were blocked with 5% non-fat dry milk (NFDM) in TBS-T (20 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.1% (v/v) Tween-20) for at least 1 h at RT. Primary antibodies were diluted in 5% NFDM in TBS-T as follows: rat α-HA 3F10 (Roche Diagnostics, Mannheim, Germany) 1:2000; mouse α-PTH1R 4D2 (Thermo Fisher Scientific) 1:2000. Membranes were incubated overnight with the primary antibody at 4 °C under constant gentle agitation, followed by 3 × 10 min washes in TBS-T. Secondary antibodies, either α-rat-HRP for α-HA (Cell Signaling Technology, Danvers, MA) or α-mouse-HRP for α-PTH1R (Santa Cruz Biotechnology, Dallas, TX) were used at dilutions 1:5000 and 1:10,000, respectively, in 5% NFDM in TBS-T. Membranes were incubated for 1 h at RT followed by 3 × 10 min washes in TBS-T. Finally, membranes were soaked in enhanced chemiluminescence reagent (10 parts 0.1 M Tris-HCl pH 8.6 with 250 mg/L luminol, 1 part DMSO with 1100 mg/L p-hydroxycoumaric acid and 0.003 parts 30% H₂O₂). After 1 min, signals were detected for 5 min in the dark (Gbox, Syngene, Bangalore, IN).

To localise expressed proteins incorporating the HA tag, immunofluorescence assays were performed using methodology set out in Tonkin et al. [59 (link)], with minor modifications. When indicated, cells were incubated with 20 nM MitoTracker CMXRos (Sigma) for 30 min at 37°C prior to fixation. Cells were washed once in DPBS before fixing in a solution 4% paraformaldehyde, 0.0075% glutaraldehyde in DPBS. After one wash in DPBS, they were permeabilised in 0.1% Triton X-100/DPBS. Another wash in DPBS was performed, and cells were blocked in 3% BSA/DPBS overnight at 4°C. Primary antibodies diluted in 3% BSA/DPBS were added and incubated for 1 h. Dilutions and antibodies used were as follows: rat α-HA 3F10 (Roche, 1:500), rabbit α-HA (Abcam, 1:500), rabbit α-PfERD2 (MR4, 1:500), rat α-PfAMA1 (MR4, 1:200), rat α-PfBIP (MR4, 1:200), rabbit α-LDH (1:2000). After antibody incubation, cells were washed 3 times in DPBS for 10 min; and the secondary antibodies were added diluted 1:5000 in 3% BSA/DPBS and incubated for 1h. Cells were then washed 3 times in DPBS for 10 min and mounted with ProLong Gold Antifade Mountant with DAPI (ThermoFisher). The slides were sealed and imaged on a DMi8 (Leica Microsystem) using LasX software. Images were processed using ImageJ 1.51k software [60 ]. Our protocol is available on protocols.io [61 ].

Tachyzoites treated with IAA or vehicle for three growth cycles were inoculated onto coverslips with confluent monolayers of HFFs and grown for 24 h. Cells were fixed in 4% (vol/vol) paraformaldehyde/PBS (Sigma-Aldrich) for 10 min and permeabilised in 0.1% (vol/vol) Triton X-100/PBS before blocking in 3% (wt/vol) BSA/PBS (Sigma-Aldrich) for 1 h. Cells were incubated with primary antibodies used at 1:1,000 in 3% BSA/PBS for 1 h. Primary antibodies used were as follows: rat α-HA 3F10 (1:1,000, Cat# 11867423001, RRID:AB_390918; Roche); mouse α-SAG1 DG52 (Burg et al, 1988 (link)); rabbit α-GAP45 (1:1,000) (Gaskins et al, 2004 (link)); rabbit α-ACP (Waller et al, 1998 (link)); and rabbit α-IMC1 (Ward & Carey, 1999 (link)) (Table S7). Cells were washed 3X in PBS and probed for 1 h with Alexa Fluor–conjugated secondary antibodies (Thermo Fisher Scientific; see Table S8 for full list), plus 5 μg/ml DAPI used at 1:1,000 in 3% BSA/PBS. Coverslips were washed and mounted onto glass microscope slides with Vectashield (Vector Labs). Parasites were imaged on a Zeiss Live Cell Axio Observer widefield microscope. Images were processed in ImageJ (Schneider et al, 2012 (link)).