Sc 365189

The following antibodies were used as primary antibodies for immunofluorescence microscopy: SGO1 (SAB1405371, Sigma Aldrich), GFP (ab290, Abcam) and CENPA (07–574, Millipore; and ab13939, Abcam). For immunoblotting, the following primary antibodies were used: SA1 (ab4457, Abcam), SA2 (A300-158a, Bethyl Laboratories), SMC1 (A300-055A, Bethyl Laboratories), SCC1 (05-908, Millipore), WAPL (A-7, sc-365189, Santa Cruz), Sororin (ab192237, Abcam), HSP90 (sc-13119(F-8), Santa Cruz) and α-tubulin (T5168, Sigma Aldrich). All primary antibodies were used at a 1:1000 dilution with the exception of HSP90 and α-tubulin (1:10000). For coimmunoprecipitation, we used 4.5 μg of SMC1 (A300-055A, Bethyl Laboratories) or IgG (2729 S, Cell Signaling) per sample. Secondary antibodies were used at a 1:1000 dilution. For immunofluorescence microscopy we used: Alexa Fluor 488 goat anti-mouse (A-11001, Life Technology), Alexa Fluor 568 goat anti-mouse (A-11004, Life Technology), Alexa Fluor 488 goat anti-rabbit (A-11008, Life Technology) and Alexa Fluor 568 goat anti-rabbit (A-11011, Life Technology). For western blots, we used the following secondary antibodies: anti-goat-PO (P0449, DAKO), anti-rabbit-PO (P0448, DAKO) and anti-mouse-PO (P0447, DAKO).

Cell lysates were prepared by a lysis buffer (20 mM Tris-HCl [pH 7.4], 135 mM NaCl, 1.5 mM MgCl₂, Triton 1%, glycerol 10%, and 1× protease inhibitor [Roche]). Immunoblots were probed with antibodies against Wapl (Sc-365189; Santa Cruz) and alpha-tubulin (T0674; Sigma-Aldrich) and developed using goat anti-mouse IgG horseradish peroxidase (HRP) conjugated antibody (Sc-2005; Santa Cruz) in a Chemidoc (GE Healthcare).

Primary antibodies used in this study: γ-H2AX (1:1000, 05-636, Millipore), TRF1 (1:500, sc-56807, Santa Cruz), TRF2 (1:1000, NB110-57130, Novus Biologicals), WAPL (1:1000, SC-365189, Santa Cruz), β-actin (1:10,000, A5441, Sigma), BAX (1:1000, rat monoclonal 49F9; WEHI monoclonal antibody facility), BAK (1:2000, rabbit polyclonal B5897, Sigma), CldU (1:25, 6326, Abcam), IdU (1:5, 347580, BD Biosciences), p53 (p53 (DO-1): 1:200, sc-126, Santacruz), p53-ser10 (1:1000, 9284S, Cell Signalling), Vinculin (1:10,000, V9131, Sigma). Secondary antibodies used in this study: Alexa Fluor 568-Goat anti-mouse IgG (1:1000, A-11031, Thermo Fisher Scientific), Alexa Fluor 488-Goat anti-mouse (1:1000, A11029, Thermo Fisher Scientific) and Alexa Flour 594-Goat anti-rat IgG (1:1000, A-11007). Goat anti-Rabbit HRP (1:1000, P0448, Dako), Goat anti-Mouse HRP (1:1000, P0447, Dako).