Fig. 5: PfCERLI2 is peripherally associated with the cytosolic face of the rhoptry bulb membrane.
a PfCERLI2HAGlmS Schizont lysates were treated with either saponin alone, saponin and digitonin, or saponin, digitonin, and proteinase K. Saponin lyses the RBC membrane and PVM, while digitonin lyses the parasite plasma membrane but leaves the membrane of internal organelles intact. Treated lysates were then probed with a cocktail of anti-HA (PfCERLI2) and anti-GAP45 (inner-membrane complex, exposed to cytosol) antibodies (outlined in blue) or RAP1 (rhoptry bulb lumen) antibodies (outlined in yellow). Antibodies against RAP1 and GAP45 serving as negative and positive controls, respectively, for proteinase K digestion. Images representative of 3 independent experiments. RBC = RBC membrane, PVM = PV membrane, PM = parasite plasma membrane, IMC = inner-membrane complex, OM = organellar membrane, OL = organelle lumen. b PfCERLI2HAGlmS schizont lysates were subjected to differential lysis and solubilisation to determine the membrane solubility of PfCERLI2. Lysates were first hypotonically lysed, before being treated sequentially with sodium carbonate and triton-x-100. The supernatant was collected following each treatment, along with a final triton-x-100 soluble fraction. Each fraction was then probed with anti-HA (PfCERLI2), anti-GAPDH (cytosolic), anti-RH5 (peripheral) and anti-MSP1 (GPI-anchored) antibodies. Images representative of 3 independent experiments.
