(c) L1502 (5
0 -AGTCAACAGATTATTGCCGATG-3
0 ).
(d) L1503 (5
0 -TACAAATCCTAATGAATTGCTTAC-3
0 ).
2.7.2 Analysis
of the p36p
À Parasite
Phenotype During Blood
Stage and Mosquito Stage
Development
1. Swiss mice.
2. Anopheles stephensi.
3. 300 μl of PBS (see Subheading 2.3.1, item 6) (see Note 1).
4. Cell counters.
2.7.3 Analysis
of Characteristics
of the Infectivity of p36p
À
Sporozoites
1. BALB/c and C57BL6 females of weight 15–20 g.
2. 5 Â 10
4 purified sporozoites.
3. Giemsa stain.
4. Cell counter.
5. Centrifuge.
6. Glass coverslips.
7. 0.02% gelatin in water.
8. Anti-CS 3D11 antibody (Ab).
9. HepG2 cells.
10. MEM medium [22].
11. Rhodamine-dextran (1 mg/ml).
12. Anti-PbEXP-1.
13. Anti-HSP90 or anti-HSP70 [23].
14. DAPI.
2.7.4 In Vitro Analysis
of Apoptosis in RAS
and p36p
À
Parasite-Invaded
Hepatocytes
1. 3 Â 10
4 sporozoites (WT, p36p
À , or RAS).
2. Gamma source, 16 Krad [24].
3. 2 Â 10
5 HepG2 cells.
4. DMEM.
5. 10% FCS.
6. 1% penicillin/streptomycin.
7. 1 mM glutamine.
8. Anti-HSP70 Ab [23].
9. Active caspase-3 detection kit (Promega).
10. DAPI stain.
2.7.5 In Vivo Analysis
of Apoptosis in p36p
À
Parasite-Invaded
Hepatocytes
1. C57BL/6 mice.
2. 5 Â 10
5 sporozoites of p36p
À or WT.
3. Anti-HSP70.
4. DAPI stain.
340
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