FIG 6.

Phos-tag separation followed by Western blotting to determine phosphorylation of RsbR1 in different genetic backgrounds. WT, ΔrsbX, ΔrsbX + rsbX, ΔrsbR1, RsbR1_T175A, and RsbT_N49A strains were grown in BHI at 37°C until an OD₆₀₀ of ∼0.4 was reached. At this time point, protein was extracted, and the migration and level of RsbR1 were determined by Western blotting using anti-RsbR1 antibodies. Purified His-tagged protein was used as a positive control, the ΔrsbR1 mutant was used as a RsbR1 negative control, the RsbR1_T175A mutant is lacking one phosphorylation site, and the RsbT_N49A mutant is a kinase mutant, abolishing RsbR1 phosphorylation. n = 3.