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Blood, Vol. 113, Issue 4, 919-928, January 22, 2009
Functional alteration of red blood cells by a megadalton protein of Plasmodium falciparum
Blood Glenister et al.
113: 919
Supplemental materials for: Glenister et al
Files in this Data Supplement:
- Figure S1. Generation of transgenic clones (JPG, 33.3 KB)
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(A) To generate Pf332 KO clones, 3D7 was transfected on two separate occasions with the KO construct pHTKΔ332. Following each transfection, parasites were cultured separately. Following both positive and negative drug selection, transgenic parasites were single-cell cloned by limiting dilution to generate four KO clones. (B) To generate Pf332 truncation mutant clones, 3D7 was transfected with either pHC1-332Δa or pHC1-332Δb. Drug resistant transformants were cultured and subjected to multiple rounds of drug cycling to eliminate episomal plasmid. Single-cell cloning led to the generation of three clones for each transfected line. Following genotypic analysis, one of these clones (for each construct) was cloned again by limiting dilution to give rise to a further three sub-clones. Two of the clones (circled), one from the first round and one from the second round of limiting dilution, were selected for analysis.
- Figure S2. RT-PCR confirmed transcription of Pf332 is completely ablated in Pf332 KO parasite clones (JPG, 33.6 KB)
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cDNA from parental 3D7 parasites or Pf332 knockout clones (11G7, 12F3, 13E2 and 13F7) was generated using a Pf332 specific primer or oligo-dT respectively, and subjected to PCR using primers at the extreme 5′ end of the Pf332 gene (RT+). A product of the expected size was observed for 3D7 parasites, whereas no product was detected for any of the four knockout clones. Control reactions using P. falciparum genomic DNA as template (gDNA), no DNA or with reverse transcriptase omitted (RT−) are shown.
- Figure S3. Abundance of proteins in membrane skeleton fractions from RBCs infected with parental or mutant parasite clones appears similar (JPG, 46.1 KB)
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Silver-stained SDS-PAGE analysis of Triton X-100-insoluble/SDS-soluble membrane fractions from uninfected RBCs (RBC) or iRBCs (3D7, Pf332 knockout clones 11G7, 12F3, 13E2, and 13F7 or truncation mutants 3F3 and 12H9, 5G1, and 13F5) showing no obvious differences in the overall abundance of RBC membrane skeleton or skeleton-associated proteins between parental 3D7 parasites and any of the mutant parasite clones.
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