Sc 1496

Primary antibodies against RORα (ab60134), E-cadherin (ab40772), vimentin (ab92547), Snail (ab53519), matrix metalloproteinase-9 (MMP-9) (ab38898), tissue inhibitor of metalloproteinase-3 (TIMP-3) (ab39184), Ki-67 (ab66155), and CD34 (ab81289) were provided by Abcam (Cambridge, MA, UK). Primary antibodies targeting β-catenin (sc-1496), p-β-catenin (sc-101650), Axin-1 (sc-14029), c-Jun (sc-44), c-Myc (sc-40), TCF-4 (sc-271287), and β-actin (sc-8432) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-conjugated secondary antibodies were provided by Abzoom (Dallas, TX, USA). Goat anti-rabbit IgG-HRP (KGAA35) and goat anti-mouse IgG-HRP (KGAA37) were provided by KeyGEN BioTECH Corp (Jiangsu, China). Goat anti-mouse IgG (H + L) was purchased from Protech Technology, Inc. (Rocky Hill, NJ, USA). pGL3-c-Myc promoter luciferase reporter plasmid was obtained from Guangzhou Cyagen Biosciences Inc.

After surgical resection, each tumor specimen was immediately formalin-fixed and then paraffin-embedded for routine and immunohistochemical investigation. For immunohistochemistry staining, 3 μm-thick sections were taken on poly-l-Lysine coated slides. Briefly, the polyclonal antibody to β catenin (sc-1496) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was incubated overnight with tissue sections according to the kit’s instructions. The peptide was used to generate preabsorption to assess the staining specificity of β-catenin. The pattern of staining observed with the polyclonal antibody was confirmed on duplicate slides using a monoclonal antibody (sc-7963) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). For the negative control, the primary antibody was substituted for PBS.

The protein extracted from the tissues and cells was resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Hybond-P; cat no. 10600088; GE Healthcare, Buckinghamshire, UK) followed by western blot analysis. Signals were detected using an enhanced chemiluminescence detection system (ECL Plus Western Blotting Detection system; GE Healthcare). Primary goat anti-SFRP2 antibody was purchased from Sigma-Aldrich (cat: SAB2500934; Shanghai, China). Goat antibodies against glycogen synthase kinase 3β (GSK-3β; sc-8257) and β-catenin (sc-1496), and rabbit antibodies against cyclin D1 (sc-753) and β-actin (sc-130656) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The poly-HRP anti-goat IgG was purchased from Abcam (ab6741; Cambridge, MA, USA). The goat anti-rabbit IgG (NA934) were purchased from GE Healthcare.