As shown in Fig 5B, the addition of NAC prevented OHT induced-activation of caspase-3

3 does have orthologs in trypanosomes. Interestingly, the conserved protein kinase domain of isoforms 1 and 2 are more similar to mammalian casein kinases than to CK1.4. The conserved protein kinase domain of L. infantum CK1.2 shows 69% identity over 295 amino acids to Mus musculus CK1 epsilon, but only 32% identity over 310 amino acids to LdCK1.4. LdCK1.4 was analyzed using SecretomeP version 2.0 and SignalP version 3.0, programs that predict non-classical and classical protein secretion. The former program gives a SecP score = 0.7959, while the latter program predicts a short 13 amino acid signal peptide region with a protease cleavage site between amino acids 13 and 14. 9. Analysis of CK1.4 Secretion by Ld:CK1.4-FLAG Mutants Induced release of CK activity was carried out essentially as described with the following modifications. Ld:CK1.4-FLAG or Ld:wt parasites collected at 0, 3, 5, 10, and 15 min. Western blot analysis was carried out as described above in section 6. 10. Mutant and Wild-type Promastigote Growth in Culture Promastigotes, Ld:wt and Ld:CK1.4-FLAG, were diluted in complete culture media containing 10% Alamar blue solution and aliquoted in sterile 96-well flat bottom plates. The plates were incubated at 26uC, and the fluorescence was read daily over 6 days using 12411425 a fluorescent microplate reader. Promastigote growth was also measured by counting live parasites daily in a Neubauer haemocytometer. All experiments were performed in triplicates. Results were analyzed using Prism 6. 11. Analysis of Differentiation into Metacyclic Stage Promastigotes Differentiation in culture of Ld:wt, Ld:CK1.4-FLAG and Ld:LUC parasites into metacyclic stage promastigotes over 7 days was determined by flow cytometry. Samples were removed, washed 15722457 and adjusted to 1.56106 cells/ml in ice cold PBS containing 10% FCS and 1% sodium azide. The cells were stained with propidium iodide for 5 min, washed by centrifugation with FACS buffer, and finally suspended in FACS buffer. Forward and side scatter parameters were collected with a flow cytometer and analyzed using Summitv4.3 software. Correct gating of procyclic and metacyclic promastigote populations was determined by separation on Ficoll step gradients, as previously described, and analysis by flow cytometry. 12. Infection of Mouse Macrophages by Mutant and Wild-type Promastigotes Resident peritoneal macrophages were isolated from thioglycollate stimulated BALB/c mice and allowed to adhere overnight to Lab-Tek II 8well chamber slides. Non-adherent cells were removed by washing with warm medium and the macrophages infected for 3 hrs in quadruplicate with either Ld:wt, Ld:CK1.4-FLAG or Ld:LUC stationary phase promastigotes. Excess parasites were removed by washing 3 times with warm medium and the slides BHI1 further incubated for 72 hrs. Slides were removed, stained with Diff-Quick, and the % infected macrophages and number of parasites per infected macrophage determined by light microscopy. 2. Expression and Activity of Recombinant His-tag LdCK1.4 Polypeptides Full-length and three deletion constructs were cloned into pET28a, and the polypeptides expressed in E. coli by induction with IPTG and arabinose. Western blotting analysis detected major bands representing each His-tagged CK1.4 polypeptide at appropriate molecular weight in lysates from the induced bacteria. The predicted molecular mass of each recombinant polypeptide is: full-length LdCK1.4, 62 kDa; LdCK1.4D190, 51 kDa; LdCK1.4411566, 45 kDa; and

评论已关闭。