Mouse anti v5 r960 25

S2 and HEK293T cells were lysed with HEPES lysis buffer (150 mM NaCl, 50 mM HEPES pH 7.5, 0.5% (v/v) Triton X-100 supplemented with protease inhibitor cocktail with EDTA (Roche) and the soluble fraction was obtained by centrifugation (16,000 × g, 20 min). For co-IP experiments cleared lysates were added to 20 μL of ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich) or 15 μL of GFP-Trap agarose (ChromoTek) in 100 μL HEPES lysis buffer. The lysate and the beads were incubated for 1 h at 4 °C and washed four times with HEPES lysis buffer. Input and co-IP samples were analysed by SDS-PAGE and western blot. Primary antibodies were: mouse anti-FLAG (M2), 1/5000 (Sigma-Aldrich F1804); mouse anti-GFP (3E1), 1/1000 (Cancer Research UK); rat anti-HA (3F10), 1/2000 (Roche); mouse anti-Myc (9E10), 1/5000 (Santa Cruz sc:40); mouse anti-Tubulin (E7), 1/5000 (Developmental Studies Hybridoma Bank); mouse anti-V5 (R960-25), 1/5000 (Thermo Fisher), rabbit anti-P-TAZ^Ser89 1/1000 (E1X9C) (Cell Signalling Technologies 59971), rabbit anti-HA 1/1000 (C29F4) (Cell Signalling Technologies 3724). Secondary antibodies were from GE Healthcare (1:5000).