LACK Gene Expression Analysis in L. major

Genomic DNA from the virulent LACK/LACKL. major control line as well as the LACK/LACK^RDG and LACK/LACK^DDE lines was extracted with phenol-chloroform (34 (link)), digested overnight with StuI, concentrated by ethanol precipitation, and size separated by agarose gel electrophoresis. The DNA was transferred onto Hybond-N+ nylon membrane (GE Healthcare Life Sciences, Piscataway, NJ) by alkali capillary blotting following the manufacturer’s instructions. A biotin-labeled probe, derived from the LACK ORF, was synthesized using a NEBlot Phototope kit (New England BioLabs, Ipswich, MA) and purified using a QIAquick nucleotide removal kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s recommendations. The labeled probe was hybridized to the target DNA at 65°C using Rapid-hyb buffer (GE Healthcare Life Sciences) according to the manufacturer’s instructions. Following hybridization, the membrane was washed in 2× saline sodium citrate (SSC) plus 0.5% SDS followed by 0.2× SSC plus 0.5% SDS as previously reported (7 (link)). The blot was visualized by chemiluminescence using the Phototope-Star detection kit (New England BioLabs) as per the manufacturer’s specifications.