Samples were pelleted and resuspended in Laemmli buffer containing 5% 2-mercaptoethanol and stored at −20 °C. Proteins were separated on 4–20% Tris–HCl SDS-PAGE TGX gels in running

buffer (25 mM Tris base, 192 mM glycine, 10% SDS). Frozen lysates were boiled for 5 min and held on ice for 5 min before use. The RC DC Protein Assay was performed to equalize the amount of total protein loaded in each lane. All protein supplies were obtained from Bio-Rad unless otherwise stated. Proteins were transferred to an Immun-Blot PVDF membrane using a Trans-Blot apparatus. The membrane was blocked overnight at 4 °C in 0.05% Tween 20 in Tris-buffered saline (TBS) containing 5% nonfat dry milk on a Belly Dancer. Primary antibodies used at 1 : 10 000 dilutions were either an antipeptide Selleck Buparlisib antibodies directed against amino acids 5–19 of UmuDAb or polyclonal antibody prepared by GenScript by injection of purified UmuDAb XAV-939 (produced by GenScript) into rabbits and purified by protein A chromatography. Goat anti-rabbit HRP-conjugated secondary antibody was used at a dilution of 1 : 32 000. All antibody incubations were carried out for 1 h in 0.05% TBS Tween 20

in 2.5% milk on a Belly Dancer. Precision StrepTactin-HRP Conjugate was added with the secondary antibody to visualize the protein size marker (Precision Plus Protein WesternC Standards). The membrane was washed five times (10 min each) with 0.01% TBS Tween 20 after each antibody incubation. SuperSignal West Pico chemiluminescent substrate (Pierce) was used to visualize

proteins after exposure to X-ray film. UmuDAb expression and cleavage was investigated after transforming E. coli AB1157 wild-type and mutant cells with plasmids bearing various umuDAb alleles. This allowed us to test the effects of recA and umuD mutations on UmuDAb cleavage in a context of the otherwise intact and well-studied DNA damage response of E. coli. Escherichia coli cells were exposed to DNA-damaging agents, and immunoblot analyses of cell lysates were performed with anti-UmuDAb peptide or polyclonal antibodies. To test whether the umuDAb ORF truly encoded an extra-large UmuDAb protein, plasmid pJH1, which contains 2.2 kbp of DNA from ADP1, including umuDAb in its native chromosomal context, was used as a UmuDAb expression source. This approach was feasible 4��8C because Acinetobacter promoters are typically highly expressed in E. coli (Shanley et al., 1986). Lysates from E. coli wild-type and ΔumuD cells, carrying pJH1 but not treated with MMC, expressed a c. 24-kDa protein (Fig. 2), consistent with the predicted molecular weight of 23.4 kDa, and demonstrating that the protein encoded by umuDAb was indeed larger than the 15-kDa UmuD (Kitagawa et al., 1985). This protein was not expressed in cells containing only the pUC19 vector of pJH1. This UmuDAb expression in uninduced E. coli may be due to the lack of an E.