Protein Isolation and Immunoblotting Analysis

Cells for isolation of total proteins were harvested at the same time-points and from the same cultures as that used for RNA preparation (Northern blot analysis described above). One milliliter aliquots were harvested and kept at −80 °C, until all samples were collected. The cell pellets were thawed on ice, suspended in 50 mM TrisHCl, pH = 7.4, to a calculated OD₆₀₀ of 10.0. PMSF, Dnase, Rnase, and lysostaphin were added to the samples, and they were incubated at room temperature for 15 min. Cellular debris was removed by centrifugation and the protein concentration of each sample was measured using the Bradford dye-binding procedure from Bio-Rad. For immunoblotting, a total of 5 μg of each sample was loaded on NuPAGE^® 4–12% Bis-Tris gels (Invitrogen), using MOPS-Buffer (Invitrogen). The proteins were blotted onto a polyvinylidene difluoride membrane, using the XCell II blotting module (Invitrogen). The membranes were pre-blocked with IgG, before probing with antibodies directed against the Rot transcriptional regulator (Rabbit-anti-Rot-antibody [37 (link)]) at a 1:2000 dilution. Bound antibody was detected with the WesternBreeze Chemiluminescent Anti-Rabbit kit or Anti-mouse kit (Invitrogen). The Western blot analysis was repeated three times with similar results.