Fig. 9

Identification of SNAP50 as part of a heavy protein complex that specifically binds to the Pr77 sequence. a The binding reaction of 100,000 ³²P-dsPr77 and 3 μg of nuclear protein (NP) of the parasite was performed by incubation at 37 °C for 30 min. The same binding reactions were subjected to UV-crosslinking (+) at 3000 W/cm² and 254 nm and to heat denaturing (HD+) by heating at 95 °C for 5 min. A total of 100,000 cpm ³²P-dsPr77 probe was included as a control. Products were electrophoresed on a 6% polyacrylamide native gel, transferred to PVDF membranes and visualized by PhosphorImager exposure. The black arrowheads indicate the formed complexes of reduced motility, and the asterisk indicates the electrophoretic mobility of the ³²P-dsPr77 free conformation. b The binding reaction was performed by incubating 8 pmol (~ 400 ng) of cold dsPr77 and 60 μg of nuclear protein (NP) of T. cruzi at 37 °C for 30 min. The same reactions were subjected to UV-crosslinking (+) and heat denaturing (HD+) by heating at 95 °C for 5 min. Fifty micrograms of nuclear protein (NP) of T. cruzi was included as a control. Products were electrophoresed on a 6% polyacrylamide native gel, transferred to PVDF membranes and analysed by western blotting using an anti-SNAP50 specific antibody (dil. 1:5000)