Western blotting

Proteins were extracted from lung adenocarcinoma cell lines with lysis buffer (250 mM Tris‐HCl pH 6.8, 2% SDS, 10% glycerol, 1% β‐mercaptoethanol, 2 mM phenylmethylsulphonyl fluoride). Five micrograms of each protein sample were applied to a 10% polyacrylamide gel for SDS polyacrylamide gel electrophoresis. Separated proteins on polyacrylamide gels were transferred to 0.45 μm polyvinylidene difluoride membranes (Merck‐Millipore, Darmstadt, Germany) overnight with a constant voltage of 10 V with transfer buffer (100 mM Tris (hydroxymethyl) aminomethane, 200 mM glycine). After blocking with 0.5% casein for one hour, membranes were incubated with nondiluted hybridoma supernatant containing the anti‐IMMT antibody for two hours at RT and HRP‐conjugated anti‐mouse IgG antibody (Dako, Glostrup, Denmark) diluted 1000‐fold for 45 minutes at RT. After each incubation step, the membranes were washed three times with Tris (hydroxymethyl) aminomethane‐buffered saline (TBS) containing 0.1% Tween 20 (TBS‐T) for 5 minutes each. Immunoreactive bands on the membranes were developed with Immobilon Western Chemiluminescent HRP Substrate (Merck‐Millipore) and captured with the Cool Saver System (ATTO, Tokyo, Japan).