FIG. 1.
Immunoblot of P. mirabilis whole-cell extracts with FlhD and FlhC antisera (upper panels). The lowest panel is a corresponding Northern blot of total mRNA with an flhDC probe (7). Cells were recovered from 1.5% agar plates at 0.5-h intervals spanning the early, peak, and postdifferentiation periods. Aliquots were resuspended in loading buffer (2% SDS, 50 mM Tris-HCl [pH 6.8], 100 mM dithiothreitol), and equivalent amounts of protein extract were separated by SDS–15% PAGE and electrotransferred to nitrocellulose filters. Immunoblots with affinity-purified FlhC and FlhD antisera were developed with horseradish peroxidase-conjugated goat anti-rabbit antibody and a chemiluminescent substrate (Pierce). Antisera were raised (Scottish Antibody Production) against His-tagged proteins generated from pET15b plasmids (Novagen) in E. coli BL21 (DE3) (27) carrying the flhD::Tn10 mutation (19) transferred by P1 transduction.