Fig. 7. Detection of flagellin gene mRNA by qRT-PCR and protein stability by turnover assays.

(A) The levels of flaA, flaB1, flaB2, and flaB3 transcripts were measured by qRT-PCR as previously described (Bian et al., 2013). The dnaK gene transcript was used as an internal control to normalize the qRT-PCR data. The results are expressed as Tde960^mut flagellin gene transcript expression relative to the wild-type levels. Asterisks indicate that the difference between the wild-type and Tde960^mut transcript levels was statistically significant at a P value of < 0.01 (two-way ANOVA). (B) Protein translation was arrested by adding spectinomycin to the cultures of the wild-type and Tde960^mut strains. Samples were withdrawn at the indicated time points and analyzed by immunoblotting. The whole cell lysates of the wild-type (2.5 μg) and Tde960^mut strains (20 μg) were separated by SDS-PAGE, then transferred to PVDF membranes, and finally probed with antibodies against DnaK, FlaA, or FlaBs. DnaK was used as a sample loading control. SuperSignal West Femto Maximum Sensitivity substrate was used to develop the immunoblots of Tde960^mut.