Sc 2032

Protein was extracted from cells harvested two days after transfection with the indicated siRNAs using NETN buffer (20 mM Tris-HCl pH 8, 420 mM NaCl, 1 mM EDTA, 0.5% Igepal CA-630, 1 mM DTT, and Roche Protease Inhibitor Cocktail) and 8 freeze/thaw cycles. Blots (20–50 μg of total protein) were probed with the following antibodies: BARD1 (Bethyl, A300-263A; Santa Cruz Biotech, Sc11438), BRCA1 (Abcam, ab16780), 53BP1 (Abcam, ab36823), Flag M2-HRP (Sigma, A8592), Phospho RPA32 S4/S8 (Bethyl, A300-245A), BRCA2 (EMD Millipore, OP95-100UG), RAD51 (Santa Cruz Biotech, sc-8349), Actin (Abcam, ab3280), Tubulin (Santa Cruz Biotech, sc-53030), HA.11 (16B12) (Covance, MMS-101P), or GST-HRP (NEB, E2624S) according to the instructions provided by the manufacturers. If needed, the blots were incubated with HRP-conjugated secondary antibodies (Pierce 31450 for rabbit anti-mouse IgG-HRP; Sigma A6154 for goat anti-rabbit IgG-HRP; Santa Cruz Biotech Sc-2032 for goat anti-rat IgG-HRP) before visualization of protein signals using the ECL kit (Thermo Scientific Pierce).

30 mouse metaphase I oocytes per lane were washed in BSA-free M2 medium and re-suspended in 4 µl of BSA-free M2 medium. 12 µl of 1.333× NuPAGE LDS sample buffer (Thermo Fisher Scientific) with 100 mM DTT was then added, and the mixture was immediately snap-frozen in liquid nitrogen for 5 min. Samples were thawed and frozen twice more before heated at 100°C for 5 min. Samples were resolved on a 15-well NuPAGE 4 - 12% bis-tris protein gel of 1.0 mm thickness (Thermo Fisher Scientific) with NuPAGE MOPS SDS Running Buffer (Thermo Fisher Scientific). Proteins were transferred onto a 0.45 µm PVDF membrane with SDS-free Towbin buffer at 200 mA for 2 h on ice. Blocking and antibody incubations were performed in tris-buffered saline (TBS) with 5% skim milk and 0.1% tween-20. Primary antibodies used were mouse anti-AURA (610938; BD Biosciences), rabbit anti-chTOG, mouse anti-CHC17 rabbit anti-TACC3 and rat-α-tubulin. Secondary antibodies used were HRP-conjugated anti-mouse (P0447; Dako), anti-rabbit (31462; Invitrogen) and anti-rat (sc-2032; Santa Cruz Biotechnology). Blots were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and documented with Amersham Imager 600 (GE Healthcare). Care was taken that the exposure time did not cause saturation.