Western blot analysis.

E. coli cells carrying the indicated plasmids were grown at 37°C to an A₆₀₀ of 0.3 to 0.5 and induced by the addition of appropriate inducers for 30 min. One milliliter of each cell culture was removed and the cells collected by centrifugation at 16,000 × g for 2 min at room temperature. The cell pellet was resuspended in 10 μl of 1× SDS loading buffer (10% SDS, 10 mM 2-mercaptoethanol, 20% glycerol, 0.05% bromophenol blue, 0.2 M Tris-HCl [pH 6.8]) and boiled for 5 min. Ten microliters of proteins in the sample was separated by electrophoresis on a 10% polyacrylamide–SDS gel. Proteins were transferred onto a nitrocellulose membrane (Bio-Rad) by electroblotting at 90 mA overnight at 4°C. The proteins were detected using anti-GFP monoclonal antibody (1:1,000; Roche) for sfGFP-tagged protein. The primary antibody was detected with anti-mouse IgG (goat) antibody (1:5,000; PerkinElmer) and developed using a Western Lightning Plus-ECL system (PerkinElmer). GroEL served as a loading control for E. coli and was detected by anti-GroEL antibody (1:10,000; Sigma-Aldrich) and anti-rabbit IgG (goat) secondary antibody (1:5,000; PerkinElmer) and developed using the Western Lightning Plus-ECL system (PerkinElmer).