(a)
(b)
(c)
(d)
Figure 5: CK1.1 is nonessential in L. donovani. (a) PCR analysis of the ΔCK1.1 cell line. Diagrams showing the CK1.1 locus and PCR primers (small arrows) used to test for the presence of the CK1.1 CDS or the correct integration of puromycin and blasticidin drug-resistance genes (top panel). PCR products run on an agarose gel to assess the correct integration of the puromycin-resistance gene (P), blasticidin-resistance gene (B), and the presence/absence of the CK1.1 CDS (bottom panel). Fragments sizes in kb are indicated on the left. (b) Promastigotes were seeded at promastigotes/mL and were cultured for 8 days. Aliquots were taken every 24 h to assess cell number (black symbol) and percentage of death (white symbol) by flow cytometry in triplicate from two independent experiments. Cell lines: LdB pTB007 (circle), LdB pTB007 ΔCK1.1 (diamond). (c) Proteins were extracted from LdB pTB007 or LdB pTB007 ΔCK1.1 promastigotes in logarithmic phase (right panel) and axenic amastigotes 48 h after temperature and pH shift (left panel) and twenty micrograms was analysed by Western blotting using an anti-CK1.2 antibody (Top panel). The Coomassie-stained membrane of the blot is included as a loading control (bottom panel). Protein weight is in kDa is indicated on the left. (d) Similar to (b), except that promastigotes were seeded at promastigotes/mL, shifted to 37°C and pH 5.5 and cultured for 6 days.