Figure_6.tif (489.04 kB)

In vivo cross-linking experiments.

Download (0 kB)
posted on 2014-02-27, 03:48 authored by Daniela Scribano, Andrea Petrucca, Monica Pompili, Cecilia Ambrosi, Elena Bruni, Carlo Zagaglia, Gianni Prosseda, Lucia Nencioni, Mariassunta Casalino, Fabio Polticelli, Mauro Nicoletti

The 43PPPP46 motif of PhoN2 is not required for the PhoN2-OmpA interaction. Cross-linking of HND115 (pHND10) was achieved by treating bacteria with formaldehyde to a final concentration of 1%, as described in Materials and Methods. Samples were suspended in Laemmli buffer and either heated at 37°C for 10 min to maintain cross-links or at 95°C for 20 min to break cross-links. Equal amounts of proteins were analyzed by Western blot. A protein molecular weight marker (Pierce) was used to determine the molecular weight of proteins. Immunoblotting was carried out with monoclonal anti-HA (Panels A and B) or polyclonal anti-OmpA antibodies (Panels C and D). Expression of PhoN2-HA was achieved by growing bacteria in the presence of 0.016% of L-arabinose. Panels A and C, bacteria not induced with L-arabinose; Panels B and D, L-arabinose induced bacteria. OmpA (U), unfolded OmpA; OmpA (F), folded OmpA [48]. Experiments were repeated at least three times and typical results are shown.