Excision efficiency at the protein level was assessed by WB analysis. Synchronous parasite cultures were DMSO or RAP treated as described above, and parasite pellets collected at schizont or merozoite stage. Soluble parasite lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane using an Appleton Woods wet blotter apparatus. Blots were blocked with 5% BSA (Sigma) in PBST (1% Tween 20 in PBSA) for 2h, followed by 1-h incubation with a 1:1000 dilution of anti-HA antibody (Anti-HA 3F10 monoclonal rat antibody, Roche) in 2% BSA and 3 washes with PBST. The blot was then incubated for 1h with a 1:10,000 dilution of anti-rat-HRP (Goat, polyclonal, anti-rat-HRP, Bio-Rad) in 2% BSA, followed by three washes in PBST. HRP was activated by adding Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized using a ChemiDoc Imager (Bio-Rad).
WB analysis of APEH and LYPLA1 was performed as above using 1:500 dilutions of anti-LYPLA1 (Abcam) or anti-APEH (Atlas Antibodies) monoclonal antibodies for 1h. After washing, this was followed by 30 min incubation with anti-rabbit-biotin (Goat, polyclonal, Novex), and finally 30 min with 1/2000 Streptavidin-HRP (Invitrogen).
WB analysis of APEH and LYPLA1 was performed as above using 1:500 dilutions of anti-LYPLA1 (Abcam) or anti-APEH (Atlas Antibodies) monoclonal antibodies for 1h. After washing, this was followed by 30 min incubation with anti-rabbit-biotin (Goat, polyclonal, Novex), and finally 30 min with 1/2000 Streptavidin-HRP (Invitrogen).