Anti myc antibody sc 40

Binding of FOCAD to HSP90AB1 was verified using 6xHis-tag-mediated pull down as detailed in Supplementary material online resource. Binding of FOCAD to tubulin isoforms was assayed using Myc-tag-mediated pull down. HEK293T cells were transiently cotransfected with constructs expressing N-terminal Myc-tagged TUBA1A, TUBB3, TUBB6 or TUBG1 and untagged FOCAD. At 24 h post transfection, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed for 30 min on ice in 1 ml of immunoprecipitation (IP) lysis buffer (20 mM Tris-HCl, pH 8.0), 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1% Triton X-100, supplemented with protease and phosphatase inhibitors (Roche Diagnostics)). Cell lysates were incubated with 1 µg of anti-Myc antibody (sc-40; Santa Cruz Biotechnology, Dallas, TX, USA) by tumbling over night at 4°C. Protein G Sepharose beads (GE Healthcare, Little Chalfont, UK) were equilibrated in IP lysis buffer, blocked with 0.1% bovine serum albumin (BSA; Thermo Fisher Scientific) and incubated with the lysates for 4 h at 4°C. After five washing steps with IP lysis buffer, bound protein was eluted from the beads using Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 1 mM EDTA, 0.01% bromophenol blue), and detected by Western blot analysis.