Rabbit anti trf2

Whole protein extracts were obtained by treating cell pellets with lysis buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.5 mM EGTA, 0.25% NP-40, 1 mM DTT, 0.5 mM PMSF, 0.5 mM Na₃VO₄ and protease inhibitor cocktail, Roche). Samples were loaded onto pre-cast 4–12% gradient acrylamide gels (NuPage, Invitrogen). After electro-blotting, filters were incubated for 1 h with mouse monoclonal anti-AKTIP (1 : 400, Sigma), goat anti-actin-HRP conjugated (1 : 3500, Santa Cruz), mouse anti-lamin A/C (1 : 200, Santa Cruz), goat anti-lamin B1 (1 : 200, Santa Cruz), mouse anti-FLAG-HRP (1 : 1000; Sigma), rabbit anti-matrin 3 (1 : 1000, Santa Cruz), goat anti-importin 7 (1 : 200, Santa Cruz), mouse anti-trimethyl H3K9me3 (1 : 350, Millipore) and rabbit anti-TRF2 (1 : 3500, Novus Biologicals). Filters were then incubated with appropriate HRP-conjugated secondary antibodies (Santa Cruz; diluted according to the manufacturer's instructions), which were detected using the enhanced chemiluminescence system (ECL Plus, Amersham). Bands were imaged with the ChemiDoc MP imager (Bio-Rad) and band intensities were quantified using the Image Lab software (Bio-Rad).

Cells were resuspended in Laemmli buffer and boiled for 5 minutes. Samples were loaded and electrophoresed through a SDS-polyacrylamide gel. Samples were transferred onto PVDF membranes and processed for western blot using rabbit anti-TRF2 (Novus Biologicals), mouse anti-tubulin (Sigma-Aldrich) and rabbit anti-p85 antibodies (Abcam). Peroxidase-labeled goat anti-rabbit IgG and peroxidase-labeled goat anti-mouse IgG were used as secondary antibodies. The Bio-Rad Clarity ECL reagent was used for detection.