Clh104ap

Parasites were synchronized twice at an 18-hour interval so that the remaining parasites in culture were at the late ring stage (between 18 and 22 hours), with a 0.3 M alanine-10mM HEPES solution (as described in^{47 (link)}). Synchronous parasites were then harvested by saponin lysis, the pellets solubilised in SDS protein sample buffer and separated on a 7.5% SDS-PAGE gel under reducing conditions and transferred to PVDF membranes (Millipore). The antibodies (rabbit serum anti-GAP50 1:2000^{48 (link)}, mouse monoclonal anti-HA, (Cedarlane, 1:2000, CLH104AP) were diluted in 0.1% (v/v) Tween 20-phosphate-buffered saline with 1% (w/v) skim milk. Appropriate HRP-coupled secondary antibodies were used and immunoblots were revealed by ECL (Amersham Biosciences). For all expression analyses, proteins extracted from an equal number of cells were used for each time point.

Fluorescence images of parasites were captured using a GE Applied Precision Deltavision Elite microscope with 100x 1.4NA objective and with a sCMOS camera and deconvolved with the SoftWorx software. Chromatic calibration of the microscope was performed prior to imaging experiments. For immunofluorescence assays, parasites were fixed on slides using 4% paraformaldehyde (ProSciTech) (127 (link)) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). After blocking in 3% bovine serum albumin (Sigma Aldrich) the cells were incubated for 1 h with rabbit polyclonal anti-PfERD2 (1:2000) (128 (link)), mouse monoclonal anti-HA (Cedarlane, CLH104AP, 1:2000), goat anti-human Hb (1:1000, Cedarlane) or rabbit polyclonal anti-BiP (1:1000) (125 (link)). Bound antibodies were then visualized with either Alexa Fluor-594 anti-rabbit, anti-mouse or anti-goat IgG and Alexa Fluor-488 anti-mouse or anti-rabbit IgG diluted 1:1000 (Cedarlane). Parasites were mounted in Vectashield (Vecta Laboratories) containing 0.1 μg/ml 4', 6–diamidino-2-phenylindole (Dapi, Invitrogen). Images shown represent a single optical slice from a deconvolved z-stack. For the LysoTracker labeling experiment, PfPX1-GFP parasites were incubated for 2 h with 75 nM of LysoTracker Red DND-99 (Invitrogen).

For the time-course of expression analysis, parasites were synchronized twice at an 18-h interval (resulting in 18 and 22 h ring stages), with a 0.3 M alanine-10mM HEPES solution (as described in (123 (link))). Synchronous parasites were then harvested by saponin lysis, the pellets solubilized in SDS protein sample buffer and separated on a 7.5% SDS-PAGE gel under reducing conditions and transferred to PVDF membranes (Millipore). The antibodies rabbit polyclonal anti-PfHSP70 (SPC-186C; StressMarq Bioscience Inc., 1:20000) (124 (link)), rabbit polyclonal anti-BiP (1:1000) (125 (link)), rabbit polyclonal anti-plasmepsin II (126 (link)) (1: 2000), mouse monoclonal anti-GFP (Roche, JL8, 1:1000) and mouse monoclonal anti-HA (Cedarlane, CLH104AP, 1:2000), were diluted in 0.1% (v/v) Tween 20-phosphate-buffered saline with 1% (w/v) skim milk. Appropriate HRP-coupled secondary antibodies were used and immunoblots were revealed by ECL (Amersham Biosciences). For all expression analyses, proteins extracted from an equal number of cells were used for each time point.