Fig. 6: Knockdown of PfCERLI2 inhibits rhoptry antigen processing, but not secretion, and is associated with rhoptry malformation.
a PfCERLI2HAGlmS ring-stage parasites were GLCN treated (+) or left untreated (−), before enzyme treatment of RBCs to prevent reinvasion. Lysates (P) and culture supernatants (SN) were harvested following schizont rupture and probed with antibody cocktails according to the colour theme: anti-RH4, anti-RON4 (rhoptry neck), anti-aldolase (loading control) antibodies (outlined in purple), anti-EBA-175 (microneme), anti-RAP1 (rhoptry bulb), anti-aldolase (loading control) antibodies (outlined in blue), anti-HA (PfCERLI2), anti-aldolase (loading control) antibodies (outlined in yellow). Representative blots of 5 biological replicates shown. b Western blots were normalised to the loading control, aldolase, and quantified, with results displayed as protein expression in GLCN treated sample as a percentage of the untreated signal. n = 5 biological replicates. c Quantification of individual band intensities, as a percentage of the total signal, from RAP1 signals using the parasite lysates from the secretion assay. n = 5 biological replicates. ns = p > 0.05, ****p < 0.0001 by two-way ANOVA. d Transmission electron microscopy of C1 schizont arrested PfCERLI2 and 3D7 WT parasites, either in the presence or absence of GLCN. Representative images from 3 biological replicates. Scale bar = 500 nm. e Quantification of rhoptry length from transmission electron microscopy images. n = 3 biological replicates. ***p < 0.001 by unpaired t-test. All error bars = SEM.
