Luminograph 3

Colorectal cancer cell lysates were prepared in RIPA lysis buffer (Thermo Fisher Scientific, MA, USA) with protease inhibitor cocktail (Gendepot, Barker, TX, USA). Cell lysates and culture supernatant samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. Protein-transferred membranes were blocked with 5% skim milk in Tris-buffered saline Tween 20 (TBS-T) blocking buffer for 1 h at room temperature and incubated at 4 °C overnight with blocking buffer-diluted anti-CCSP-2 IgG (1:1000) and anti-CCSP-2 scFv (1:1000) as primary antibodies. Anti-mouse IgG-HRP (1:2000; Cell Signaling Technology, MA, USA) for anti-CCSP-2 IgG and anti-human kappa IgG-HRP (1:2000; Novus Biologicals, Littleton, CO, USA) for scFv antibodies were used as secondary antibodies. Anti-β-actin antibody (Sigma-Aldrich Co., MO, USA) was used as the loading control. Protein expression signals were detected with ECL substrate (Thermo Fisher Scientific, MA, USA) and visualized using Luminograph III (ATTO Corporation, Tokyo, Japan).

The cell lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Inc.) and protease inhibitor cocktail (GenDEPOT, Barker, TX, USA). Cell lysates were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Protein-transferred membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline Tween 20 (TBS-T) for 1 h at room temperature and incubated with blocking buffer-diluted primary antibodies, including E-cadherin (Abcam; ab76055), iNOS (Abcam; ab178945), c-Met (Abcam; ab51067), VEGFA (Abcam; ab46154), Ki-67 (Abcam; ab16667) and ABCB1 (Abcam; ab170904) antibody (1:1000) at 4°C overnight. After the membranes were washed and incubated with secondary antibodies, immunoreactive protein expression signals were detected using the enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific, Inc.) and visualized using Luminograph III (ATTO Corporation, Tokyo, Japan). The expression of anti-β-actin (Sigma-Aldrich Co.) was used as the loading control.