Rabbit anti ha

The transductants (1 × 10⁶) were washed with PBS, lysed with 100 μl of lysis buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin) and incubated on ice for 10 min. After centrifugation at 21,500 g at 4°C for 10 min, the supernatant was mixed with Laemmli's sample buffer and boiled. The lysate was resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). After the membrane was blocked either with 5% skim milk (Wako Pure Chemical Industries, Osaka, Japan) for the detection of the HA tag, V5 tag and β-tubulin or with 3% bovine serum albumin (Sigma) for the detection of p-c-kit Y721. The blot was probed with rabbit primary antibodies, followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Biosource, Camarillo, CA, USA). The primary antibodies used are: rabbit p-c-kit Y721 (which corresponds to Y719 of mouse origin) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-HA (BETHYL, Montgomery, TX, USA), rabbit anti-V5 (Millipore, Billerica, MA, USA) and rabbit anti-β-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Detection was performed using Luminata Western HRP Substrates (Millipore).