Figure 2

Verification of the TPx-2 null phenotype in gene-disrupted P. berghei. WT, TPx-2 WT, and TPx-2 KO parasite populations were inoculated into mice, and parasite-infected erythrocytes, total protein, and total RNA of the parasite cells were prepared [15]. (A) The absence of TPx-2-specific mRNA expression in TPx-2 KO was examined by RT-PCR analysis. Total RNA samples (1 μg) were reverse transcribed and the cDNAs were amplified with the sequence-specific primer pairs that amplify pbtpx-1 (PBANKA_130280) and pbtpx-2 (PBANKA_143080). The primers used were: 5^′-CGG AAT TCA TGC CAT CAA TTG TAG GAA ATC AAG CC-3^′ and 5^′-GCG GAT CCT TAC AAA CTC GAT AAA TAT TTT TGC AAC TCC-3^′ for pbtpx-1, and 5′-GGA TCC TCT CAT GTT ACT CAG AAG GC-3^′ and 5^′-TAG CCT CGA GTT ACT TTT TGT ATT C-3^′ for pbtpx-2, respectively. Amplification of pbtpx-1 but not pbtpx-2 from TPx-2 KO cDNAs indicates the absence of TPx-2-specific mRNA expression in this population. Molecular size markers in bp are indicated on the left. (B) The absence of TPx-2 protein in TPx-2 KO was examined by Western blot analysis [15]. Total protein samples (5 μg) together with recombinant TPx-2 protein (rTPx-2; 5 ng) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE; 12.5%) and probed with anti-recombinant PbTPx-2 (rPbTPx-2) rabbit serum (1:1000). Anti-rPbTPx-2 rabbit serum was prepared as previously described [16] except that pGEX-6P-1 (Amersham Biosciences) was used. Protein size markers in kDa are indicated on the left.