Horseradish peroxidase conjugated rabbit anti mouse or goat anti rabbit secondary antibodies

Western blot assays were conducted as described previously [7 (link)]. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% or 10% gels (Thermo) and were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). PVDF membranes were blocked in 1 mg/mL BSA (Sigma-Aldrich) at room temperature for 1 h. Primary antibodies were diluted 1:2000 and incubated overnight at 4 °C. Secondary antibodies diluted 1:5000 were added and incubated at room temperature for 1 h. The band intensities corresponding to the protein samples were measured using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The primary antibodies used were as follows: mouse anti-β-catenin, mouse anti-lamin A/C (BD Biosciences, CA, USA), mouse anti-TCF4 clone 6H5-3 (Millipore), mouse anti-N-EBP1 (C11) (Santa Cruz, CA, USA), rabbit anti-EBP1, rabbit anti-histone H3, rabbit anti-α-tubulin, rabbit anti-HIF-1α and rabbit anti-β-actin (GeneTex, CA, USA). Horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology) were used as appropriate.

Assays for real-time PCR and immunoblotting were conducted as described previously [53 (link),54 (link)]. The primers used in real-time PCR are shown in Supplementary Table S2. The following primary antibodies were used in the immunoblotting assay: anti-NKX6.1 (AF5857; R&D systems, Minneapolis, MN, USA), anti-E-cadherin (610405; BD Biosciences, San Jose, CA, USA), anti-vimentin (sc-6260; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-SNAIL (GTX100754; GeneTex, Irvine, CA, USA), anti-TWIST (ab49254; Abcam, Cambridge, MA, USA), and anti-SLUG (#9585; Cell Signaling, Danvers, MA, USA). Horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as appropriate.