FIG. 3.

Western blotting analysis of RadA and a subunit of the RFC complex. Experiments were performed with early-stationary-phase “P. abyssi” cells after the cells were irradiated at 2,500 Gy (+γ) or not irradiated (−γ). Culture samples were removed every 30 min (up to 2 h at 96°C) and kept on ice. The soluble proteins (supernatant fraction [S]) and the DNA-bound proteins (pellet fraction [P]) were extracted from the cells as described earlier (19). Proteins were subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and were analyzed by Western blotting. The RadA (38.4 kDa) and RFC-S (37.4 kDa) proteins were immunodetected by specific rabbit antibodies by using the ECL Western blotting kit (Amersham Pharmacia Biotech). The protein band recognized by specific antibodies was visualized by exposure of the membranes to film.