Primary rabbit polyclonal antibodies

The content of β-catenin, phospho-β-catenin (Thr41/Ser45), and Axin2 in cellular extracts was assessed using the Western blot technique. Cytosolic (β-catenin, phospho-β-catenin, Axin2) or nuclear (β-catenin) extracts were separated on 7.5% SDS-PAGE gels (Bio-Rad, USA) and transferred onto nitrocellulose membrane. After blocking with 10% skimmed milk, the membranes were incubated with primary rabbit polyclonal antibodies (Santa Cruz Biotechnology, USA) directed against β-catenin, phospho-β-catenin or Axin2. The analysis of β-actin or lamin A served as a loading control. After washing, the membranes were probed with alkaline phosphatase-labeled secondary antibodies (anti-rabbit IgG, Santa Cruz Biotechnology, USA) and stained using the BCIP/NBT AP Conjugate Substrate Kit (Bio-Rad, USA). The Quantity One software was used to determine the amount of the immunoreactive products and the values were calculated as relative absorbance units (RQ) per mg protein.

Total protein was extracted from mouse cecum tissue using TRIzol Reagent (Life Technologies, Grand Island, NY). 15 μg of total protein was resolved using 12.5% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 2% nonfat dried milk and incubated at 4°C with primary antibodies. Detection of mouse HSP90 α/β was performed with primary rabbit polyclonal antibodies (Santa Cruz Biotechnology), while detection of S100A9 was performed with polyclonal goat anti-mouse S100A9 (R&D Systems). Lcn-2 was detected by polyclonal goat anti-mouse Lcn2 (R&D Systems). After overnight incubation at 4°C, the blots were washed and then incubated for 1 h at room temperature with secondary goat anti-rabbit and donkey anti-goat antibodies conjugate to horseradish peroxidase (HRP) (Southern Biotech and Santa Cruz Biotechnology, respectively). After washing, bands were developed using the SuperSignal West Pico Chemiluminescent Sustrate (Thermo Scientific) per manufacturer’s instructions and visualized using Gel Doc (BioRad).