C3956

Reagents were purchased from Sigma-Aldrich unless noted otherwise. Restriction endonucleases, T4 ligase and calf intestinal alkaline phosphatase were from New England Biolabs. RNA was isolated using the Pure Link RNA Mini kit from Ambion and transcribed into first-strand cDNA using SuperScript III first-strand cDNA synthesis kit (Invitrogen). Phusion high-fidelity polymerase, dNTPs, DreamTaq DNA polymerase, GeneRuler DNA ladders, PageRuler plus prestained protein ladder, HisPur cobalt resin and the enhanced chemiluminescence (ECL) system for western blot were from Thermo Scientific. All oligonucleotides used in this study are listed in Supplementary Table 1 and were synthetized by IDT or LifeTechnologies (Invitrogen). Reagents for cell culture were from Biowest. The pG140 and pG152 vectors were provided by Markus Meissner (University of Glasgow, UK). The following primary antibodies were used in western blot or immunofluorescence assays: mouse or rabbit anti c-myc (monoclonal antibodies, M4439 and C3956, Sigma), anti-PentaHis antibody (Qiagen), rabbit anti-TgHsp90 (Echeverria et al., 2010 (link)) kindly provided by Sergio Angel (National University of San Martín, Argentina) and anti-TgDHO (Robles Lopez et al., 2006 (link)).

Western blot analyses for Col-0; P_FT-FT-FM-3′ UTR, Col-0; P_FT-FT-FM-pri-miR159a-3′ UTR, and Col-0; P_FT-FT-FM-pri-miR159a-T1-3′ UTR were performed as described previously [70 (link)]. The blots were detected with antibodies against Myc (Sigma-Aldrich, C3956) and actin (Sigma-Aldrich, A0480). Secondary antibodies were goat-developed anti-rabbit (GE Healthcare, NA934) and anti-mouse immunoglobulin G (GE Healthcare, NA931). Western blot membranes were developed with ECL+, and signals were detected with ChemiDoc XRS+ and captured with the Image Lab software (Bio-Rad) in accordance with the manufacturer’s instructions.

Mice were perfused transcardially with PBS followed by 4% paraformaldehyde prepared in PBS. Brains were post-fixed 1 h at 4°C and immunohistochemistry was performed on cryosections (20 μm) encompassing the entire visual cortex. Primary antibodies included anti-Myc (rabbit, 1/400, Sigma-Aldrich C3956), anti-Otx2 (mouse monoclonal, in house), anti-PV (rabbit, 1/500, Swant PV25), anti-CR (mouse monoclonal, 1/500, Swant), anti-GABA (rabbit, 1/300, Sigma), and anti-cFos (rabbit monoclonal, 1/300, Cell Signaling). Secondary antibodies were Alexa Fluor-conjugated (Molecular Probes). Biotinylated WFA (1/100, Sigma-Aldrich L1516) with Alexa Fluor-conjugated streptavidin was used to reveal perineuronal nets. Sections were mounted in Fluoromount (Southern Biotech). Images were acquired with a Leica SP5 confocal microscope and analyzed with ImageJ.

Immunofluorescence, Western blot, and flow cytometry analysis were performed using antibodies against GFP (1:1,000, 11814460001, Roche; or 1:1,000, ab290, Abcam), PARP1 (1:1,000, 9542, Cell Signaling, Alexis), Myc (1:1,000, 9E10, SC-40, Santa Cruz), γH2AX (1:1,000, 07-164, Millipore), α-tubulin (Sigma-Aldrich), DNA-PKcs (1:500, ab1832, Abcam), LIG4 (1:1,000, 80514, Abcam), XRCC4 (1:500, gift from Mauro Modesti, Marseille Cancer Research Center, Marseille, France), histone H3 (1:2,000, 1791, Abcam), GST (1:2,000, Amersham), PARP1 (1:1,000, 9542S, Cell Signaling), PARP2 (1:500, C3956, Sigma-Aldrich), ZBTB24 (1:1,000, PM085, MBL), CDCA7 (1:250, ProteinTech), RAD51 (1:2,000, sc-6862, Santa Cruz), CD4-FITC (1:100, 100509, BioLegend), β-actin (1:2,000, AC15, Sigma-Aldrich), PAR (1:1,000, 4336-BPC-100, Trevigen; used in Fig. 5, A and B), PAR monoclonal 10H, which was purified from the culture medium of 10H hybridoma obtained from Dr. Miwa (Nagahama Institute of Bio-Science and Technology, Nagahama, Japan) through the Riken cell ban (Kawamitsu et al., 1984 (link)), and custom-made monoclonal AID (Jeevan-Raj et al., 2011 (link)).

SDS-PAGE and BN-PAGE gels were blotted onto BioTrace™ PVDF membranes (Merck Millipore, Carrigtwohill, Ireland). Electrophoretic transfer of proteins was performed for 90 min at 30 V, 170 mA in the case of SDS-PAGE gels, or for 60 min at 25 V, 130 mA in the case of BN-PAGE gels. After transferring, in the case of BN-PAGE, the membranes were fixed in 8% acetic acid solution for 15 min at room temperature and rinsed with deionized water. Membranes were blocked with 5% milk in PBS overnight at room temperature, then rinsed with 0.05% Tween in PBS. Membranes were subjected to immunoblotting using either a rabbit polyclonal antiserum raised against P66 (D8713, diluted to 1:10,000), or a polyclonal rabbit anti-c-Myc antibody (C3956, Sigma, Saint Louis, MO, USA) diluted to 1:10,000 (for SDS-PAGE blots) or to 1:500 (for BN-PAGE blots) in 2.5% milk in PBS. Membranes were then washed three times for 5 min each with 0.05% Tween in PBS and incubated with an anti-rabbit secondary antibody coupled to horseradish peroxidase (ECL™ anti-rabbit IgG, Horseradish peroxidase linked whole antibody, Sigma). Membranes were washed three times for 5 min each with 0.05% Tween in PBS and bound antibodies were detected using Amersham ECL™ Prime Western Blotting detection reagents (GE Healthcare, Buckinghamshire, UK).