Img 124a

At 48 h after transfection with siRNAs, adherent cells were fixed by incubation for 10 min in 4% paraformaldehyde and were then permeabilized by incubation with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 min at room temperature. For blocking, cells were incubated for 1 h at room temperature in PBS containing 7.5% goat serum and 7.5% foetal bovine serum and then incubated overnight at 4°C with the indicated primary antibody. The following antibodies were used for IF: GFP (1/200, ab6673, abcam), PML (1:100, sc966, Santa Cruz), PCAF (1:50, ab12188, Abcam), GCN5 (1:100, 3305, Cell signaling), TRF2 (1:100, IMG-124A, Imgenex), and USP22 (ab4812, Abcam). Secondary labelling was performed using an Alexa Fluor 488- or 594-conjugated antibody (Molecular Probes) at room temperature for 1 h.

IF experiments were carried out as previously described (Nandakumar et al., 2012 (link)), using the following antibodies: mouse monoclonal anti-TRF2 (IMG-124A; 1:500; Imgenex), rabbit polyclonal anti-coilin (sc-32860; 1:100; Santa Cruz, Dallas, TX), rat monoclonal anti-mCherry (M11217; 1:1000; Life Technologies), and rabbit polyclonal anti-RAP1 (NB-100-292; 1:500; Novus Biologicals, Littleton, CO). Secondary antibodies (Life Technologies, Abcam) were pre-absorbed to prevent cross-reactivity between rat and mouse antibodies.

Exponentially growing cells were lyzed with 500 μL per 60mm dish of TNE buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1% Triton X100) supplemented with phosphatase inhibitors (10 mM NaF, 2 μM Na3VO4, 20 mM β-glycerophosphate) and a protease inhibitor cocktail (Roche Diagnostic). Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C. Immunoprecipitated proteins were visualized with anti-TRF2 or anti-PX[phospho]SP antibodies respectively. The anti-phosphorylated forms of TRF2 were obtained as the following: anti-phosphopeptide sera were generated by Eurogentec (Liege, Belgium) by injecting two rabbits each with the following phosphopeptide. NH₂- LPA -(PO₃H₂)-SPALKNKR-COOH coupled to KLH (the boldface underlined “S” represents the serine targeted by ERK1/2). Sera were affinity-purified by passing them first over an EAH-Sepharose 4B column (Amersham Biosciences, Inc.) to which the unphosphorylated peptide was coupled and the flow-through was collected. The non-retained fraction was then passed over a column to which the phosphorylated peptide was bound. Specific IgG were then eluted with 100 mM glycine (pH 2.8) and neutralized in Tris 3 mM, pH 11.

Cells were fixed in 2% paraformaldehyde in PBS for 10 min at RT, permeabilized in 0.5% NP-40/PBS for 10 min at RT, blocked in 1% BSA/PBS, and incubated with mouse anti-Myc clone 4A6 (1.0 µg/ml; Milipore #05-724) and rabbit anti-tankyrase 1 762 (1.4 µg/ml)^{57 (link)}, mouse anti-γH2AX clone JBW301 (1 µg/ml; Millipore #05-636) and rabbit anti-53BP1 (4 µg/ml; Novus Biologicals #NB100-304) or rabbit anti-TIN2 701 (0.36 µg/ml)^{58 (link)}, or rabbit anti-TRF1 415 (0.2 µg/ml)^{59 (link)} and human anti-centromere (ACA) (1:4000) antibodies. For Rad51 and TRF2 coimmunofluorescence, cells were permeabilized in Triton X-100 buffer (0.5% Triton X-100, 20 mM Hepes-KOH at pH 7.9, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose) for 5 min at RT, fixed in 3% paraformaldehyde (in PBS, 2% sucrose) for 10 min at RT, permeabilized in Triton X-100 buffer for 10 min at RT, blocked in 1% BSA/PBS, and incubated with rabbit anti-RAD51 (4 µg/mL; Santa Cruz #sc-8349) and mouse anti-TRF2 (2.5 µg/ml, Imgenex #IMG-124A). Primary antibodies were incubated at RT for 2 h, followed by detection with FITC-conjugated or TRITC-conjugated donkey anti-rabbit or antimouse antibodies (1:100; Jackson Laboratories #711-025-152 or #711-095-152). DNA was stained with 0.2 µg/ml DAPI.