Anti mouse ig hrp

The following antibodies were used for western blot: rabbit polyclonal anti-XLF (Bethyl, Montgomery, TX, USA; A300-730A, dilution 1:2000), swine polyclonal anti-rabbit immunoglobulin-horseradish peroxidase-conjugated (Ig-HRP; Dako antibodies, Dako, Glostrup, Denmark; #P0399, dilution 1:5000), mouse monoclonal anti-β-actin (Abcam, Cambridge, UK; ab8226, dilution 1:2000), rabbit polyclonal anti-mouse Ig-HRP (Dako antibodies, Dako, Glostrup, Denmark; #P0260, dilution 1:5000), and goat polyclonal anti-mouse Ig-HRP (Dako antibodies, Dako, Glostrup, Denmark; #P0447, dilution 1:5000).

The region corresponding to target RNAs selected according to the RIPseq experiments was amplified from bacterial DNA adding a T7 promoter at the 5' end. PCR products were used in MEGAshortscript T7 Kit (Ambion) to produce in vitro RNA (S6 Table). 2μM of Biotin-11-CTP was added into the reaction mix for later detection. This reaction mix was incubated at 37°C for 2h and the RNA was purified by Phenol/Chloroform extraction. The RNA concentration was estimated by UV absorption at 260nm. For 10μl interaction assays, 200nM of RNA was combined with varying concentrations of purified CsrA-His (0–5μM) and incubated in buffer A in presence of 250ng tRNA yeast (Invitrogen) for 30min at RT. Subsequently, samples were fractionated under non-denaturing conditions on Blue-Native PAGE and blotted to BrightStar-Plus transfer membranes (Ambion). Membranes were blocked in PBS buffer containing 0.1% Tween-20 and 1% ECL blocking agent (GE Healthcare) for 1h at RT and, afterwards, incubated for 1h in the same buffer including mouse anti-biotin antibodies (Invitrogen). After washing and binding of secondary antibodies (anti-mouse Ig-HRP, Dako), the RNA-bands were visualized with ECL Plus Western blotting solutions (GE Healthcare) and detected with a G-box (Syngene).

Antibodies used for western blot (WB) include rabbit polyclonal anti‐PAXX (C9orf142; 1 : 1000 dilution; Novus Biologicals, Centennial, CO, USA; NBP1‐94172), anti‐XLF (1 : 1000; Cell Signaling, 2854), anti‐LIG4 (1 : 1000; Abcam, Cambridge, UK, ab193353), anti‐H2AX (1 : 5000; Abcam, ab11175); mouse monoclonal anti‐DNA‐PKcs (1 : 1000; Invitrogen, MA5‐13404), anti‐XRCC4 (1 : 2000; Novus Biologicals, NBP1‐48053), anti‐β‐actin (1 : 2000; Abcam, ab8226); swine polyclonal anti‐rabbit Ig‐HRP (1 : 5000; Dako, Santa Clara, CA, USA; P0399); goat polyclonal anti‐mouse Ig‐HRP (1 : 5000; Dako, P0447); IRDye® 800CW Goat anti‐Rabbit IgG (H + L; LI‐COR, P/N 926‐32211); and IRDye® 680RD Goat anti‐Mouse IgG (H + L; LI‐COR, P/N 926‐68070).

To detect the PAXX protein by western blot, we used rabbit polyclonal anti‐PAXX/C9orf142 IgG (NovusBio, Littleton, CO, USA, NBP1‐94172, dilution 1 : 500), which recognizes the C‐terminal half of the PAXX protein (amino acids 109‐204); anti‐PAXX/C9orf142 IgG (Abcam, Cambridge, UK, ab126353, 1 : 200) and swine polyclonal anti‐rabbit Ig‐HRP (Dako antibodies, #P0399, 1 : 3000). Anti‐GAPDH rabbit polyclonal (Sigma, St. Louis, MO, USA, #G9545, developed to recognize 314–333 amino acids of mouse GAPDH, 1 : 2000) and mouse monoclonal anti‐β‐actin (Abcam, ab8226, 1 : 3000) with rabbit polyclonal anti‐mouse Ig‐HRP (Dako antibodies, #P0260, 1 : 3000) were used to control protein loading.

For western blot (WB), we used the following antibodies: rabbit polyclonal anti‐PAXX/C9orf142 (NovusBio, Littleton, CO, USA; NBP1‐94172, dilution 1 : 1000); rabbit polyclonal anti‐XLF (Bethyl, A300‐730A, 1 : 2000); swine polyclonal anti‐rabbit Ig‐HRP (Dako antibodies, Dako, Glostrup, Denmark; #P0399, 1 : 5000); mouse monoclonal anti‐XRCC4 (NovusBio, NBP1‐48053, 1 : 2000); mouse monoclonal anti‐β‐actin (Abcam, Cambridge, UK; ab8226, 1 : 2000); rabbit polyclonal anti‐mouse Ig‐HRP (Dako antibodies, #P0260, 1 : 5000); goat polyclonal anti‐mouse Ig‐HRP (Dako antibodies, #P0447, 1 : 5000); rat monoclonal anti‐AID (Active Motif, Carlsbad, CA, USA; #39886, 1 : 500) and secondary anti‐rat IgG, HRP‐linked (Cell signaling #7077, Cell Signaling, Leiden, The Netherlands; 1 : 5000); rabbit polyclonal anti‐UNG (custom‐made against murine UNG, 2 μg·mL⁻¹); and IRDye 800CW anti‐rabbit IgG (Licor, Lincoln, NE, USA; #925‐32211, 1 : 15 000).

Microtiter plates were coated with 50 μl, 2 μg/ml recombinant proteins in glycine/HCl buffer and blocked with blocking buffer. 24E9 mAb (50 μl; 3-fold dilutions starting at 10 μg/ml; 1 h; room temperature) was added, and washing was performed as described above. Bound Ab was detected with anti-mouse Ig-HRP (Dako; 1:3000 in blocking buffer; 1 h; room temperature).