Journal: PLoS ONE

Article Title: Regulation of 53BP1 Protein Stability by RNF8 and RNF168 Is Important for Efficient DNA Double-Strand Break Repair

doi: 10.1371/journal.pone.0110522

Figure Lengend Snippet: A . HeLa cells were treated with or without MG132 (20 µM) before exposure to 10 Gy X- irradiation. 4 h post-IR, cells were fixed for immunofluorescence microscopy as indicated. B . different treatments were applied to HeLa cells and immunoblot for RIF1 was done. Ctrl, no treatment (lane 1) or4 h post 10 Gy-IR (lanes 2–7). Additional treatments included caffeine (10 mM; lane 3) MG132 (20 µM; lane 4), siRNA specific for RNF8 (lane 5), siRNA specific for RNF168 (lane 6), and mixed siRNA specific for both RNF8 and RNF168 (lane 7). C . MG132 (20 µM) was included in medium 30 minutes prior to exposure to 10 Gy X-ray irradiation. At 4 h post-IR, HeLa cells were extracted in situ with cell extraction buffer on ice for 15 min (+NP40) or not extracted. Cells were fixed and stained for immunofluorescence microscopy as above.

Article Snippet: We used the following primary antibodies: anti-53BP1 (Santa Cruz, H-300, for immunoblot and immunofluorescence), anti-RAD51 (Santa Cruz, H-92), anti-γ-H2AX (Millipore, clone JBW301, for immunoblot and Immunofluorescence), anti-RIF1 (Santa Cruz, N-20, for immunoblot and Immunofluorescence), anti-RNF8 (Abnova), anti-RNF168 (Abcam), anti-α-tubulin (Sigma), anti-H4 (Millipore), anti-β-actin (Cell Signaling), anti-HA (purified from mouse ascites fluid), and anti-RHA (purified from rabbit serum).

Techniques: Irradiation, Immunofluorescence, Microscopy, Western Blot, In Situ, Extraction, Staining