Fig. 6

Knocked down and overexpression of ISP1 and ISP2 genes in L. donovani. ISP1 and ISP2 KD and OE constructs were prepared and transfected in L. donovani. After successful transfection, RNA was isolated from WT, KD and OE parasites. cDNA was prepared followed by semiquantitative RT-PCR for (a) LdISP1 and (b) LdISP2, α tubulin was taken as endogenous control. Proteins were extracted from transfected parasites. Down regulation and overexpression of LdISP1 and LdISP2 was further checked at protein level by Western blot by using (c) anti-GFP antibody: Lane 1: WT parasites; Lane 2: only pLGFPN vector transfected parasites; Lane 3: ISP2KD parasites; Lane 4: ISP2OE parasites; Lane 5: ISP1KD parasites; Lane 6: ISP1OE parasites (d) anti-ISP1 antibody: Lane 1: WT parasites; Lane 2: only pLGFPN transfected parasites; Lane 3: ISP1KD parasites; Lane 4: GFP-tagged ISP1OE parasites. (e) anti-ISP2 antibody: Lane 1: WT parasites; Lane 2: only pLGFPN vector transfected parasites; Lane 3: ISP2 KD parasites; Lane 4: GFP-tagged ISP2OE parasites. α tubulin was taken as endogenous control. Densitometric analysis showed the relative fold increase in the band intensities of ISP1KD, ISP1OE and ISP2KD, ISP2OE genes or proteins as compared to WT. Experiments were performed in triplicate. Gel and blot images are representative of the single experiment. An asterisk (*) denotes P ≤ 0.05 as compared to WT