Prpa2 s33

Cells were collected in 100 to 200 μl of lysis buffer. Relative protein concentration was determined with NanoDrop and samples adjusted to the same concentration with the lysis buffer. Between 20 to 40 μl of cell lysate supplemented with 5 to 10 μl of 5x concentrated reducing loading buffer were loaded on 8 to 12 % gels (SDS page). Proteins were transferred to nitrocellulose membrane using semi-dry apparatus. Membranes were blotted with 5 % milk in PBS buffer. Primer antibodies were diluted 500 to 2000 times in PBS buffer plus 0.1 % Tween. Fluorescent secondary antibodies were applied for detection. The following antibodies were used for immunoblots: Caspase-3 (Cell Signaling, #9665), cleaved Caspase-3 (Cell Signaling, #9661), p-Chk2 T68 (Cell Signaling, #2661), p-Chk2 S33/35 (Cell Signaling, #2665), Chk2 (Cell Signaling, #2662), p-Chk1 S345 (Cell Signaling, #2341), p-Chk1 S317 (Bethyl, #A300-163A), Chk1 (Cell Signaling, #2345), p-RPA2 S33 (Bethyl, #A300-246A), RPA2 (Cell Signaling, #2208), p-H2AX (Cell Signaling, #9718), H2AX (Cell Signaling, #2595), alpha-tubulin (Sigma, #T6074-200UL).