Nbp1 90156ss

Cells were lysed in cell lysis buffer (50 mM Tris‐HCl pH 7.5, 125 mM NaCl, 5 mM EDTA and 0.1% Triton X‐100) containing both 1% protease inhibitor and 1% phosphatase inhibitor cocktail (Sigma‐Aldrich, St Louis, MO, USA). Equal amounts of protein were separated on 10% SDS‐PAGE and then transferred to polyvinylidene fluoride membranes with an iBlot^®2 Gel Transfer Stack (Thermo Fisher Scientific). The membranes were incubated with primary antibodies at 4°C overnight. The primary rabbit antibodies used were anti‐ANXA10 antibody (1:500, NBP1‐90156SS, Novus Biologicals), anti‐phosphorylated Akt (Ser473) (/1:500, #4060, Cell Signaling Technology, Beverly, MA), anti‐phosphorylated Akt (Thr308) (1:500, #2965, Cell Signaling), anti‐Akt (1:1000, #9272, Cell Signaling), anti‐phosphorylated Erk1/2 antibody (Thr202/Tyr204) (1:500, #9101, Cell Signaling), anti‐Erk1/2 antibody (1:1000, #9102, Cell Signaling), anti‐β‐actin antibody (1:1000, #4970, Cell Signaling). The membranes were then probed with secondary antibodies for 90 min at room temperature. The secondary antibody was horseradish peroxidase‐conjugated donkey anti‐rabbit IgG (1:1000, NA934V, GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Immunohistochemistry was performed on 4‐µm sections of paraffin‐embedded specimens. After deparaffinization and hydration, antigen retrieval was conducted. The endogenous peroxidase activity was quenched by 15 min in 3% hydrogen peroxide and then 15 min in bovine serum albumin at room temperature. The tissue sections were then incubated with anti‐ANXA10 rabbit polyclonal antibody (1∶200, NBP190156SS, Novus Biologicals, Littleton, CO, USA), anti‐CYB1B1 rabbit polyclonal antibody (1:50, sc‐32882, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti‐KLK6 mouse monoclonal antibody (1:50, sc‐374564, Santa Cruz) at 4°C in a moist chamber overnight. The next day, the slides were incubated with second antibodies (Envision Dual Link; DakoCytomation, Glostrup, Denmark) for 30 minutes at room temperature. The slides were then colored with 3,3'‐diaminobenzidine tetrahydrochloride (Muto Pure Chemicals, Tokyo, Japan) for 15 min. ANXA10 immunoreactivity in ESCC tissue was scored based on the intensity of positive cancer cells seen in the invasive area compared with that of the corresponding normal esophageal epithelium as a negative internal control. The staining intensity of ANXA10 was evaluated as low (negative to weak immunoreaction) and high (moderate to intense immunoreactivity). The evaluation and scoring were performed by three independent pathologists (authors H.K., Y.K. and H.Y.).