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Fig. 4 | Cell & Bioscience

Fig. 4

From: Universal minicircle sequence binding protein of Leishmania donovani regulates pathogenicity by controlling expression of cytochrome-b

Fig. 4

CBRL over-expression induces UMSBP oxidation and kDNA loss whereas TXN co-over-expression in CBRL-OE parasite counter balances the effect of CBRL in vivo. AF Confirmation of CBRL and TXN over-expression in CB+ and CB+TXN+. A, D mRNA level of CBRL and TXN by semiquantitative RT-PCR. Panel A—gel images; Panel B—densitometry data. A–tub (alpha-tubulin) PCR was as loading control. B and E Western blot analysis of GFP and TXN expression in CB+ and CB+TXN+ parasites respectively and in WT parasite. Panel A—gel images; Panel B—densitometry data. C and F The presence of pLGFP-LdCBRL and pLp-NEO2-LdTXN constructs in transfected parasite was detected by PCR. Lane M—Marker, Lane 1 shows the bands obtained by PCR using pLp-NEO2 and GFP vector specific foreward primer (designed from just upstream of the start code of LdTXN and LdCBRL respectively) and LdTXN and LdCBRL specific reverse primer respectively. Lane 2 shows the band obtained by PCR using gene specific foreward and reverse primers. G Oxidation (O-UMSBP) and reduction (R-UMSBP) level of UMSBP in CB+, WT and CB+TXN+ parasites. H Level of minicircle DNA (kDNA) in CB+, WT and CB+TXN+ parasites was determined by semi-quantitative RT-PCR. I Microscopic analysis by DAPI staining to determine the kDNA loss in WT (a), CB+ (b) and CB+TXN+ (c) parasites. N Nucleus, K kDNA and a loss of kDNA. Asterisk denotes that the data are significant (p < 0.01)

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