Fig. 4.

Validation and characterization of TbPLA₁ mutant cell lines.

A. TbPLA₁ null mutants were generated in the procyclic form of T. brucei after sequential TbPLA₁ UTR-targeted homologous recombination with UTR-flanked puromycin and blasticidin resistance genes. Cell lines were analysed by Southern blot after digesting genomic DNA with EcoRV. EcoRV-digested T. brucei WT gDNA reveals one size of DNA fragment detectable by fluorescein-labelled TbPLA₁ ORF probe. In TbPLA₁ null mutant cells (Δpla1) both TbPLA₁ alleles are absent. A tetracycline (Tet)-inducible myc-tagged recombinant ectopic copy of TbPLA₁ (PLA1-myc) cloned into a phleomycin-resistant pLEW100 cassette was introduced into a different locus in WT gDNA to produce transgenic TbPLA₁ overexpression cell lines (ovexPLA1-myc), or introduced in the null mutant gDNA to produce a rescue cell line (Δpla1 rescPLA1-myc).

B. Northern blot analysis of TbPLA₁ mutants. Total RNA extracted from WT and TbPLA₁ mutant trypanosomes were hybridized with TbPLA₁ (top panels), loading controls are also presented (bottom panels).

C. Western blot analysis of cell lysates of TbPLA₁ mutants. Both native PLA₁ (theoretically 32.4 kDa) and tagged PLA1-myc (theoretically 33.9 kDa) proteins were detected by antibodies raised against the purified recombinant enzyme. * = background bands that show loading in the null mutant lanes.