Western blotting

Western blotting was performed as described previously (Afanasyeva et al, 2011). Briefly, the whole cells were prepared in a buffer containing 7 M urea, 1% Triton X-100, 100 mM DTT, 20 mM Tris–HCl, pH 8.5. Protein concentrations were determined by Bradford assay (Bio-Rad), and 50 μg protein lysate were separated per lane on either 7.5% or 12% PAGE gels then transferred to nitrocellulose membranes (Protran). Membranes were incubated with the appropriate antibodies, and the bands were visualized using the ECL system (Pierce). Images were captured with a CCD camera (Vilber Lourmat).