Fig. 8

Identification of the nuclear TBP and SNAP50 among the nuclear proteins of the parasite that specifically bind to the Pr77 sequence. a The binding reaction was performed using 100,000 cpm of ³²P-dsPr77 and 3 μg of nuclear proteins (NP) of the parasite by incubation at 37 °C for 30 min. The same reaction was subjected to UV-crosslinking (XL+). The same amount of ³²P-dsPr77 subjected (+) or not (−) to UV crosslinking (XL) was included as a control. Reactions were loaded on 10% SDS-PAGE denaturing gels, transferred to PVDF membranes and products visualized by PhosphorImager exposure. The black arrowhead indicates the formed complexes of reduced mobility, and the asterisks indicate the electrophoretic mobility of the nucleic acid-free form. b In total, 8 pmol (~ 400 ng) of cold dsPr77 probe was incubated with 60 µg of parasite nuclear proteins, and the binding reaction was subjected to UV-crosslinking (XL). Fifty micrograms of nuclear protein from the parasite was loaded as a control. Products were resolved by 10% denaturing SDS-PAGE and transferred to PVDF membranes for subsequent western blot analysis using anti-TBP antibody (dil. 1:150). c, d In total, 8 pmol (~ 400 ng) of cold dsPr77 probe was incubated with 60 µg of parasite nuclear proteins (c) or 180 µg of total proteins (d) of T. cruzi, and the binding reaction was subjected to UV-crosslinking (XL). The same amount of nuclear (c) or total proteins (d) of the parasite was loaded as a control. Products were resolved by 10% denaturing SDS-PAGE and transferred to PVDF membranes for subsequent western blot analysis using anti-SNAP50 antibody (dil. 1:5000). The black arrowheads in b–d indicate the identified products and their molecular weight in kDa