Sc 8337

Immunofluorescence staining was performed as previously described [53 (link)]. The following primary antibodies were used: anti-53BP1 (1:250, #4937, Cell Signaling Technology), anti-phospho-histone H2A.X Ser139 (0.1 μg/mL, #05-636, Millipore, Burlington, MA), tankyrase (1 μg/mL, sc-8337, Santa Cruz Biotechnology) and V5 (0.24 μg/mL, R960-25, Thermo Fisher Scientific). Alexa Fluor 594 or 488-conjugated anti-mouse or rabbit IgG (4 μg/ml, A-11032, A-11029, A-11037 and A-11034, Life Technologies) were used as secondary antibodies. Images were acquired using an Olympus IX71 microscope with a DP80 digital camera (Olympus, Tokyo, Japan) and analyzed by cellSens software (Olympus).

HeLa cells were seeded in 24-well plates on glass slides 24 h before transfection. Eighteen hours post transfection, the cells were washed once in PBS before fixation for 20 min in PBS-3% paraformaldehyde. Fixed cells were washed with PBS and quenched for 10 min with PBS-50 mM NH₄Cl. Fixed cells were then permeabilized for 10 min with PBS-0.25% Saponin. Cells were incubated overnight at 4 °C in a wet chamber with a 1:100 dilution of a mouse anti-SSSCA1 antibody (2F5, Novus Biologicals) or a 1:200 dilution of rabbit anti-TNKS1/2 antibody (sc-8337, Santa Cruz Biotechnology) or a 1:50 dilution of mouse anti-γ-tubulin (NB100–74421, Novus Biologicals) in PBS-0.25% Saponin. Cells were washed three times with PBS-0.25% Saponin and stained for 20 min in a wet chamber with 1:200 dilution of anti-mouse-AlexaFluor 594 or anti-rabbit-AlexaFluor 680 secondary antibody (Invitrogen). After three washes with PBS-0.25% Saponin, the nuclei were stained for 8 min with 2 µg/ml Hoechst 33258 (Invitrogen). Finally, cells were washed with PBS, quickly rinsed in H₂O and mounted in mowiol 4–88. Images were obtained on LSM 510 (Zeiss) microscope with a 63 × 0.55 numerical aperture oil objective.

Proteins complexes obtained after TAP were boiled once more for 3 min at 95 °C and loaded on a 12% SDS–PAGE gel. Proteins were transferred to PVDF membranes and incubated for 1 h at room temperature in TBS-0.05% Tween 20 (TBS-T)-5% BSA or -5% nonfat dry milk. The membranes were probed overnight at 4 °C with the following primary antibodies: mouse anti-SSSCA1 (1:2000, 2F5, Novus Biologicals), rabbit anti-TNKS 1/2 (1:200, sc-8337, Santa Cruz Biotechnology), rabbit anti-GFP tag (1:1000, #2555, Cell Signaling Technology), mouse anti-β-actin (1:1000, #3700, Cell Signaling Technology), rabbit anti-c-MYC (1:1000, ab32072, Abcam), mouse anti-γ-tubulin (1:1000, NB100–74421, Novus Biologicals), diluted in TBS-T-5% BSA or −5% nonfat dry milk. Primary antibodies were visualized with secondary HRP-conjugated antibodies (Cell Signaling Technology) and Chemiluminescence Blotting substrate (Roche) on a Chemidoc Imaging System (Bio-Rad).