Imgenex 124a

For immunostaining of RAP1, TRF1, TRF2 together with lamin B1, cells grown on coverslips, 24 or 48 h after transfection, were pretreated with extraction buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Trition-X100, 1 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, as described in (64 (link)), 2 min on ice before fixation in 2% PFA (10 min)). For single staining with lamin B1, cells were directly fixed in 4% PFA or in ice-cold 100% methanol (10 min). Then, cells were saturated in PBS with 2% BSA-0.05% Tween and stained for 1 h with primary antibodies (mouse anti-TRF1 (Sigma T1348), mouse anti-RAP1 (ab14404, Abcam), mouse anti-TRF2 (Imgenex 124A or Santa Cruz B5) and rabbit anti-lamin B1(ab16048, Abcam), then washed and incubated 1 h with the secondary antibodies (alexa fluor-488 (Life Technologies) or mouse primary antibody and alexa fluor -594 (Life Technologies) for rabbit primary antibody). Nuclei were then counterstained with DAPI and slides were mounted with fluoromount mounting medium (Southern Biotech). Images were acquired on epifluorescence microscope leica DM5500B using a 63×-oil objective and analyzed with ImageJ software.

Cells grown on coverslips were fixed in methanol for 10 min, blocked and stained as described above for immunostaining with the following primary antibodies couples: mouse anti-TRF2 (Imgenex 124A) with rabbit anti-lamin B1 (ab16048, Abcam) or mouse anti-RAP1 (ab14404, Abcam) with rabbit anti-lamin B1 (ab16048, Abcam) or mouse anti-HA (HA.11, Biolegend) with rabbit anti-lamin B1 (ab16048, Abcam) or mouse anti-GFP (ab1218, Abcam) with rabbit anti-lamin B1 (ab16048, Abcam) or rabbit anti-Flag (F7425, Sigma) with mouse anti-TRF2 (Santacruz B-5). PLA was performed using the Duolink in situ detection Kit (Sigma) according to the manufacturer's protocol. For some experiments, when indicated in figure legends, PLA was coupled with immunostaining of lamin B1 to detect lamin B1-positive cells. For experiments with Flag-lamin B1 constructs or with HA-TRF2-Linker, anti-Flag and anti-HA antibodies, were used to detect Flag or HA expression intensity, respectively. Digital images were acquired with the SPE confocal microscope using a 63×-objective lens. Images were processed with ImageJ software.