Polysome profiling and Western blot analysis of B. subtilis strains

Experiments were performed as described above for E. coli strains, with minor modifications.

VHB90 and VHB91 strains were pre-grown on LB plates overnight at 30 °C. Fresh individual colonies were used to inoculate 200 mL LB cultures. The cultures were grown at 37 °C until OD₆₀₀ of 0.3 and IPTG was added to final concentration of 30 μM. After 30 min, cells were collected by centrifugation (8000 rmp, 10 min), dissolved in 0.5 mL of Polymix buffer ^[108] [20 mM HEPES–KOH (pH 7.5), 95 mM KCl, 5 mM NH₄Cl, 10 mM Mg(OAc)₂, 0.5 mM CaCl₂, 8 mM putrescine, 1 mM spermidine, 1 mM DTT, 2 mM PMSF], lysed (FastPrep homogeniser (MP Biomedicals): four 20 s pulses at speed 6.0 mp/s with chilling on ice for 1 min between the cycles) and clarified by ultracentrifugation (14,800 rpm, 20 min).

Clarified cell lysates were loaded onto 7%–35% sucrose gradients in Polymix buffer ^[108] [20 mM HEPES–KOH (pH) 7.5, 95 mM KCl, 5 mM NH₄Cl, 10 mM Mg(OAc)₂, 0.5 mM CaCl₂, 8 mM putrescine, 1 mM spermidine, 1 mM DTT] and subjected to centrifugation (35,000 rpm for 3 h at 4 °C). C-terminally HTF-tagged VmlR (wild-type and EQ₂ mutant) and ribosomal protein L3 of the 50S ribosomal subunit were detected using either anti-Flag M2 primary combined with anti-mouse-HRP secondary antibodies or anti-L3 primary (a gift from Fujio Kawamura) combined with goat anti-rabbit IgG-HRP secondary antibodies, respectively. All antibodies were used at 1:10,000 dilution.