Ldccys2 is a single copy gene and is expressed only in amastigotes. (A) Southern blot hybridization of digested genomic DNA from L. (L.) chagasi (Lc) and L. (L.) donovani (Ld). 5 μg of genomic DNA was digested with restriction enzymes as mentioned in the figure and blotted onto Hybond-N membranes. The blot was probed with coding region pf Ldccys2 cDNA clone. (B) Northern blot analyses of L. (L.) chagasi total RNA. Total RNA (10 μg/lane) from promastigotes of logarithmic (lane 1), stationary (lane 2) growth phase, U937 cells (human macrophage cell line) infected with promastigotes for 96 h (lane 3) and uninfected U937 cells (lane 4) were separated on 1.2% (w/v) formaldehyde agarose gel and transferred on to Hybond N+ membrane. Blot was hybridized with PCR amplified DNA fragment containing 3'UTR from Ldccys2 (panel I), Ldccys1 (154 bp, near the polyA region, panel II), and coding region of α-tubulin from L. (L.) chagasi, a kind gift from Dr. M.E. Wilson (panel IV). (C) Western blot analysis of L. (L.) chagasi promastigotes and amastigotes. Equal amounts of proteins from promastigotes of logarithmic (lane 1), stationary (lane 2) phase, U937 cells infected with promastigotes (lane 3) and uninfected U937 cells (lane 4) were separated on 10% (w/v) SDS-PAGE and blotted on to Hybond – P membrane. Western blot analysis was carried out using α-Ldccys2 antibody.