Anti β catenin antibody

Infected IK and SHT-290 monolayers from IK/SHT-290 co-cultures were collected in 8 M urea. The protein samples were examined by SDS-PAGE electrophoresis for Sypro Ruby (BioRad) staining and Western Blot analysis as previously described (Deka et al., 2006 (link)). Blots were probed with anti-β-catenin antibodies (Cell Signaling). Protein accumulation in each experiment was quantified from triplicate samples using a BioRad G-box and SynGene software. β-catenin accumulation was normalized to the total amount of protein detected in each sample by Sypro Ruby staining.

Immunohistochemical (IHC) analysis was conducted to study protein expression in clinical specimens. The procedure was carried out similarly to previously described methods [32 (link)]. After deparaffinization, the sections were immunohistochemically stained using anti-PHF21B (HPA053834, 1:2500; Sigma-Aldrich), and anti-β-catenin antibody (#9562, 1:200; Cell Signaling). The degree of immunostaining offormalin-fixed, paraffin-embedded sections was examined and scored independently by two pathologists. The scores were determined by combining the proportion of positively stained tumor cells and the intensity of the IHC signals. Tumor cell proportions were scored as follows: 0, no positive tumor cells; 1, 1%-25% positive tumor cells; 2, 25%-50% positive cells; 3, 50%-75% positive tumor cells; and 4, >75% positive tumor cells. Staining intensity was graded according to the following criteria: 0, no staining; 1, weak staining (light yellow); 2, moderate staining (yellow brown); and 3, strong staining (brown). The staining index (SI) was calculated as the staining intensity score × the proportion of positive tumor cells (ranging from 0 to 12). We evaluated protein expression by determining the SI. Samples with an SI ≥ 6 were classified as showing high expression, while samples with an SI < 6 were classified as showing low expression.

Immunohistochemical staining for REG Iα, Ki67 and β-catenin was performed with an Envision Kit (Dako Agilent Technologies, Tokyo, Japan) as described previously [49 (link)], using anti-REG Iα antibody (dilution; 1:2000), anti-Ki67 antibody (Dako Agilent Technologies, dilution; 1:50), and anti-β-catenin antibody (Cell Signaling Technology, Danvers, MA, USA; dilution; 1:500). In brief, the rehydrated sections were treated by microwave heating for 20 min in 1×Dako REAL Target Retrieval Solution (Dako Agilent Technologies) and then preincubated with 0.3% H₂O₂ in methanol for 20 min at room temperature to quench endogenous peroxidase activity. The sections were then incubated with primary antibodies for 60 min at room temperature, washed in PBS, and incubated with horseradish peroxidase-conjugated secondary antibody for 30 min. The slides were visualized using 3,3′-diaminobenzidine tetrahydrochloride with 0.05% H₂O₂ for 3 min and then counterstained with Mayer’s hematoxylin.

Cells were seeded on the top of coverslips and cultured in serum-free conditions for 18 h, followed by incubation with media containing 10% FBS for 6 h. They were fixed with Acetone/Methanol (1:1) for 15 min at −20 °C and blocked with 1% BSA for 1 h at room temperature. Cells were incubated with an anti-β-catenin antibody (Cell Signaling) and diluted (1:100) in 1% BSA overnight, followed by incubation with Alexa Fluor^® 488 secondary antibody (Invitrogen) for 2 h. Coverslips were then incubated with 4,6-diamidino-2-phenylindole (DAPI, 1 μg/mL) for 10 min. Fluorescent signals were observed using the Zeiss LSM 700 confocal microscope (Toronto, ON, Canada).