Cd44v9

Immunohistochemistry for CD44v9 (rabbit monoclonal; Cosmo Bio) and xCT (rabbit monoclonal; Cell Signaling Technology) was carried out using the VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA, USA). Paraffin‐embedded tissue sections were deparaffinized and heated in 10 mM sodium citrate buffer (pH 6.0) at 121°C for 5 minutes for antigen retrieval. Sections were preblocked with normal horse serum from the kit and incubated with the primary antibodies at 4°C overnight. According to the manufacturer's protocol, positive signals were visualized by 0.1% 3,3′‐diaminobenzidine‐tetrahydrochloride. Cell nuclei were counterstained with hematoxylin. Samples incubated with rat or rabbit IgG were used as negative controls. Intensity of staining was scored on a scale of 0‐3 as follows: 0, negative staining; 1, mildly positive staining; 2, moderately positive staining; and 3, strongly positive staining. The signal positive area was scored on a scale of 0‐3 as follows: 0, negative staining; 1, positive staining in 1%‐10% of cells; 2, positive staining in 11%‐60% of cells; and 3, positive staining in 61%‐100% of cells. The scores were summed for each specimen. A total score of ≥1 was considered as positive expression.

In order to obtain whole cell extracts, we used RIPA buffer consisting of 50 mmol/L Tris‐HCl (pH 7.5), protease inhibitor, 1% NP‐40, 0.5% deoxycholate, 0.1% SDS, and 150 mmol/L NaCl.
In western blotting, 20 μg total protein from each sample was loaded onto 12.5% SDS‐polyacrylamide gels and transferred to a nitrocellulose membrane. The membrane was blocked at 4°C overnight in TBS containing 5% PhosphoBlocker Blocking Reagent (Cell Biolabs, San Diego, CA, USA) and 0.2% Tween‐20, and then incubated at 4°C overnight with the primary Abs for CD44v9 (1:5000 dilution; Cosmo Bio), phospho‐p38^MAPK (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA), p38^MAPK (1:1000 dilution; Cell Signaling Technology), and β‐actin (1:5000 dilution; Sigma‐Aldrich). Membranes were then washed for 5 minutes in TBS with 0.2% Tween‐20 and incubated with a peroxidase‐labeled secondary Ab (Dako, Glostrup, Denmark) for 1 hour. The membranes were washed again and analyzed after visualization using enhanced chemiluminescence reagents with the ECL Plus Western Blotting Detection System (GE Healthcare, Chicago, IL, USA). Signal intensities were quantified using the LAS 4000 system (GE Healthcare). The experiment was carried out in triplicate, and data were expressed as the mean ± SE of the relative signal intensities.