Laemmli buffer 4

Antibody purification was confirmed using SDS-PAGE. Samples were mixed with Laemmli Buffer 4× [Bio-Rad, Hercules, CA, USA, Cat. #1610747] or boiled at 95 °C for 5 mins with Reducing Buffer [Table 2] for reducing conditions then loaded into Mini-PROTEAN TGX Gels, (15-well, 15 μL) [Bio-Rad, Hercules, CA, USA, Cat. #4561036]. Gels were run at 150 V for 45 mins on a Mini-PROTEAN Tetra Vertical Electrophoresis Cell [Bio-Rad, Cat. #1658004] and visualized using InstantBlue Protein Stain [Sigma-Aldrich, St. Louis, MO, USA, Cat. ISB1L].

For analysis of protein levels, cells were grown in p60 tissue culture plates at a density of 5 × 10⁵ cell/cm², treated with iPA for 48 h and subsequently irradiate at 4 Gy. At 24 h after irradiation, cells were washed with PBS, harvested and lysed 30 min with ice-cold RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 0.5% deoxycholic acid, 10 mg/mL leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 10 mg/ml aprotinin containing protease and phosphatase inhibitors (purchased from Sigma). After being quantified using the Protein Assay Dye Reagent Concentrate (Biorad) and boiled for 5 min in Laemmli buffer 4× (Bio-Rad), equal amounts of protein samples were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (AmershamTM ProtranTM, GE Healthcare Life science, Germany). Membranes were blocked with 5% skim milk powder or 5% BSA in Tris-buffered saline containing 0,1% Tween-20 (TBST) for 1 h at RT and incubated overnight at 4°C with the specific antibodies. Next day, the membrane was incubated with appropiate secondary antibodies (Biorad) for 1 h at RT and subsequently blots were revealed by chemiluminescence (ECLTM Prime Western Blotting Detection Reagents, Amersham, GE Healthcare, Buckinghamshire, UK). Total extracts were normalized by using an anti- β-actin antibody.

For protein detection, samples of the ABE assay or 40 μg of proteins from subcellular fractionation samples were prepared with the addition of Laemmli buffer 4× (Bio-Rad, Hercules, CA, U.S.A.) and run in 12% SDS/PAGE gels. Proteins were transferred to a nitrocellulose membrane and Western blot analysis was performed using the indicated primary antibodies. Antibodies were: anti-GFP (Roche Applied Science, Penzberg, Germany) 1:1000, anti-red fluorescent protein (RFP) (Invitrogen, CA, U.S.A.) 1/1500. The blots were probed using secondary antibodies coupled to either IRdye 680 or IRdye 800 (LICOR Bioscience, Cambridge, U.K.) at 1:20000 dilution, and then scanned using an Odyssey Infrared Imager (LICOR Bioscience, Cambridge, U.K.). Quantification and statistical analysis were carried out using ImageJ and GraphPad Prism software, respectively.