FIG. 3.
Western blot analysis of BSA419 after treatment with ethanol. Cells were grown as described in the legend to Fig. 4, with samples harvested 30 min after induction by pouring over ice chips. Following centrifugation, the cells were resuspended in buffer (50 mM Tris-HCl [pH 8.0], 0.1 mM ETDA, 0.03% phenylmethylsulfonyl fluoride) and were disrupted by passage through a French press. The extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were transferred to nitrocellulose, and were probed by Western blotting by using monoclonal antibodies raised against RsbV, -W, -X, -R, -S, -T, and -U and ςB (8). The anti-RsbX antibody detects doublet bands of unknown significance (24). Lane 1, cells immediately before addition of IPTG; lane 2, 30 min after IPTG induction; lane 3, 90 min after addition of IPTG, without ethanol treatment; lane 4, 90 min after addition of IPTG, with ethanol treatment (60 min).