The mitochondria were washed two times in buffer A and pelleted

n at 10 min. The membrane was also probed with polyclonal anti-KMP 11 antibodies. KMP 11 is present in the cytoplasm and flagellar pocket, as well as on the surface and flagella of the parasite. KMP 11 was only detected in the whole parasites lysates, and not in any of the cell-free supernatants examined. This finding further indicates that the presence of CK1.4 in the cell-free supernatants is not due to cell death. 4. Localization of CK1.4 in L. donovani Nigericin (sodium salt) web promastigotes Secreted Casein Kinase 1.4 gotes were fixed with paraformaldehyde and incubated with either antibodies to the FLAG epitope, to recombinant CK1.4, or buffer alone. The fluorescence was examined after incubation with either Cy2- or Cy3 labeled secondary antibodies. The distribution of native CK1.4 in either Ld:CK1.4-FLAG or Ld:LUC parasites appears to be the same. In both parasites the fluorescence is intracellular, and present both as weak diffuse staining throughout the cytoplasm, as well as stronger punctate pattern adjacent to the nucleus and/or kinetoplast, but not co-localizing with the nuclear DAPI staining. Intensity of CK1.4 staining varies markedly between individual parasites, perhaps depending on the cell cycle. When anti-FLAG antibodies are used, the fluorescence pattern observed with Ld:CK1.4-FLAG promastigotes is essentially identical to that observed using anti-CK1.4 antibodies. Similar to the anti-CK1.4 antibodies, staining of the Ld:CK1.4-FLAG parasites with anti-FLAG antibodies gives a weak diffuse fluorescence in the cytoplasm, as well as strong punctate staining adjacent to the nucleus and/or kinetoplast. This fluorescence does not co-localize with the nuclear DAPI staining. No fluorescence was observed when anti-FLAG antibodies were incubated with Ld:LUC promastigotes or when the primary antibodies were omitted, negative controls. 5. Effect of CK1.4 Over Expression on Promastigote 1685439 Growth and Metacyclogenesis cell density than the control cultures. However by day 3, the density of the CK1.4-FLAG promastigotes was significantly higher Secreted Casein Kinase 1.4 , 9.26107 cells/ml, than either of the control cultures, and remained significantly higher on day 4 and day 7. Ld:CK1.4-FLAG promastigotes reach a density of 6.16108 cells/ml on day 7 compared to 1.6 and 1.56108 cells/ml for the Ld: wt and Ld:LUC parasites, respectively. No significance difference between the Ld:wt and Ld:LUC promastigote 14522929 growth was noted. Similar results were observed when cell growth was monitored daily using the viability indicator alamarBlue, where fluorescence is proportional to cell density. No significant difference in fluorescence between the parasite cultures was noted 24 hrs after passage. However, at 48 hrs fluorescence for parasites over expressing CK1.4 was,50% higher than the controls. All the cultures entered stationary phase by day 4, and there was little additional increase in fluorescence on day 6. On both day 4 and 6 the fluorescence was significantly higher, 76 and 81%, for the Ld:CK1.4FLAG promastigotes than the control parasites indicating that former cultures reached a higher cell density. In order to examine whether CK1.4 over expression also effects parasite differentiation in culture, we followed mutant and wildtype promastigote metacyclogenesis daily by measuring the forward-angle scatter and side-angle scatter parameters using flow cytometry. Gating of the procyclic and metacyclic promastigote populations was determined using pure parasite populations