Hrp anti rabbit 1706515

Cell pellets were lysed for 30 min in ice-cold whole cell extract buffer (50 mM TrisHCl pH 7.4, 250 mM NaCl, 0.1% Nonidet NP40, 5 mM EDTA, 50 mM NaF and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates were cleared by centrifuging at 12,000 rpm for 5 min and the protein concentration was determined using a BioRad assay kit (BioRad, Hercules, CA, USA). Cell lysates (50µg) were resolved on 10–12% SDS-PAGE (polyacrylamide gel electrophoresis) gels. Proteins were then transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). Immunoblotting was carried out with the following antibodies: rabbit anti-PARP #9542 (cell signaling, 1:1000), rabbit anti-γH2AX #9718 (Cell Signaling, Danver, MA, USA, 1:1000), goat anti-ACTIN sc-1615 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500) and mouse anti-RAN sc-271376 (Santa Cruz Biotechnology, 1:500). The secondary antibodies conjugated with horseradish peroxidase (HRP) anti-rabbit #1706515 and anti-mouse #1706516 were purchased from BIO-RAD Laboratories S.r.l., anti-goat sc-2354 from Santa Cruz Biotechnology. HRP substrate (ECL Western Blotting Detection, Amersham-Life Science, Amersham, UK) was added and the signal was detected with the Odyssey Fc instrument (Li-COR). All the uncut filter and relative densitometric raw data have been included in Figure S7.

Cells transfected or infected with control or NANEP-expressing vectors were lysed with RIPA buffer (50 mM Tris pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) supplemented with fresh protease inhibitors for 20 minutes on ice. Proteins were then analyzed on SDS-PAGE electrophoresis followed by Western Blot using specific primary antibodies anti-flag antibody: F3165 from Sigma (1:1000), anti-HA antibody C29F4 from Cell Signaling (1:1000), anti-GAPDH: 2118S from Cell Signaling (1:1000), GFP NB1001770 from Novusbio (1:1000) and appropriate HRP secondary antibodies (HRP anti mouse 170-65-16 (1:3000), HRP anti rabbit 170-6515 (1:3000), and HRP anti-goat 1721034 (1:5000) from BioRad). Chemiluminescence was determined using the ECL detection kit (GE Healthcare Amersham) and band intensities were quantified using Image Studio Lite software.