Alpha tubulin t9026

HeLa-FlpIn-TRex cells were induced with doxycyclin (2 µg ml⁻¹) 48 h before harvesting. Synchronization by thymidine (2 mM) for 24 h and release for 10 h into Taxol (2 µM) arrested cells in prometaphase. Cells were collected by mitotic shake-off. Lysis was done in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% NP40, 1 mM EDTA, 1 mM DTT, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails 2 and 3 (Sigma). Complexes were purified using GFP-Trap beads (ChromoTek) for 15 min at 4°C. Precipitated proteins were washed with lysis buffer and eluted in 5× SDS sample buffer. Primary antibodies were used at the following dilutions for western blotting: BUBR1 (A300-386A, Bethyl) 1 : 2000, alpha-tubulin (T9026, Sigma) 1 : 5000, GFP (Custom) 1 : 10 000, APC1 (A301-653A, Bethyl) 1 : 2500, APC3 (gift from Phil Hieter) 1 : 2000, MAD2 (Custom) 1 : 2000, CDC20 (A301-180A, Bethyl) 1 : 1000. Western blot signals were detected by chemiluminescence using an ImageQuant LAS 4000 (GE Healthcare) imager.

NR2F2 Isoform 1 (41859, Abcam for western blot (1:1000)); 61214, Active Motif for ChIP (5 μL per sample), NR2F2 Isoform 2 (Millipore custom antibody (1:1000)), SNAIL (3879, Cell Signaling Technology (1:1000)), Actin (A3854, Sigma (1:50,000)), Lamin-B (sc6217, Santa Cruz (1:50,000)), Alpha-tubulin (T9026, Sigma (1:50,000)). Rabbit secondary (Sigma, A0545 (1:20,000)), mouse secondary (Sigma, A9044 (1:20,000)), rat secondary (Millipore, AP136P (1:20,000)), and goat secondary (Sigma, A5420 (1:20,000)).