Fig. 5

PAR response and localization in procyclic cultures subjected to hydrogen peroxide (H₂O₂) treatment. a Analysis by Western Blot of poly(ADP-ribose) formation revealed with anti-PAR antibody (BD) after 1 mM H₂O₂ treatment for 10 min in wild type (WT), PARP over-expressing (p2216-TbPARP) and RNAi-TbPARG (p2T7-TbPARG) cultures. Data were normalized to anti-α tubulin band (LC) and are shown as the ratio of PAR to LC signals. Untreated (control) and procyclic cultures exposed to 500 μM H₂O₂ for 10 min were analyzed for PAR and PARP localization: (b) PAR was detected with specific polyclonal antibodies (BD) and nuclear PAR signal (arrow) quantification is shown below. The corrected total nucleus fluorescence (CTCF) was calculated as = Integrated Density - (Mean fluorescence of background readings X Area of selected nucleus). A Student Test was performed and significance of the nuclear signal in treated versus control parasites is indicated (*** P < 0.001). c TbPARP localization was detected with specific polyclonal antibodies (GeneScript) in untreated (control) and procyclic cultures exposed to 500 μM H₂O₂ for 10 min (arrow). d TbPARP-eYFP fusion protein localization was recognized by eYFP fluorescence and PAR localization was recognized with polyclonal anti-PAR antibody in a 3 day-induced TbPARP over-expressing cultures (p2216-TbPARP). e PAR localization was recognized with polyclonal anti-PAR antibody in a 3 day-induced RNAi-TbPARG cultures (p2T7-TbPARG). DAPI was used to identify nuclear (N) and kinetoplastid (K) DNA. White bar represents 10 μm