Sc56766

Strains of interest were harvested at mid-exponential phase (OD 0.5), lysed directly in 1X NuPAGE LDS buffer (Novex) containing 6uM DTT, separated by NuPAGE Bis-Tris gel electrophoresis and transferred onto nitrocellulose using the iBlot system (Life Technologies). Blots were incubated with rabbit polyclonal antisera against σ^E or monoclonal antibody against RpoB (sc56766, Santa Cruz Biotechnology) in 5% milk in TBST. Horseradish-peroxidase conjugated secondary antibodies (Pierce) and Supersignal West Pico chemilumeniscent substrate (Pierce) were used to detect primary antibody signal. Blots were visualized on X-ray film, which was subsequently digitized on a FujiFilm FLA-5100 imager, and bands quantitated using MultiGuage V3.1 image analysis software.

Protein samples (purified or interaction complexes) or whole-cell fractions (cell extract after sonication or cell pellets) were dissolved in Laemmli sample buffer and boiled for 5 min. The protein samples loaded onto 12% or 15% SDS-PAGE gels were separated based on molecular weights, and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dry milk in 1× Tris-buffered saline-Tween 20 (TBS-T) buffer and probed with anti-FLAG antibody (3:2,000 dilution, F1804; Sigma, St. Louis, MO, USA), anti-His₆ antibody (3:2,000 dilution, sc-8036; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-DnaK antibody (1:10,000 dilution, ADI-SPA-880; Enzo Life Science, Farmingdale, NY, USA) or anti-RpoB antibody (1:10,000 dilution, sc-56766; Santa Cruz Biotechnology, Santa Cruz, CA, USA) as primary antibodies. Anti-mouse IgG conjugated with peroxidase (3:5,000 dilution, sc-2005; Santa Cruz Biotechnology) was used as the secondary antibody in all Western blots. The chemiluminescent signals were developed with a West-Zol plus Western blot detection system (Intron Biotechnology, Seoul, South Korea).