Anti hoxa13

Tissue sections were deparaffinized, rehydrated, and heat-treated with citrate buffer (0.01 M, pH 6.0) for epitope retrieval. After exposure to 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity, the tissue sections were incubated with anti-HOXA13 (1:200; Abcam) and anti-Ki67 antibodies (1:100; Abcam) at 4° overnight. Then, the anti-mouse or anti-rabbit GTVision™ Detection System (GeneTech, Shanghai, China) was used to detect the primary antibody after incubation for 30 minutes at room temperature. After washing, the slides were counterstained with hematoxylin and mounted with coverslips. The immunohistochemical score was based on the staining intensity and the percentage of positive cells.14 (link)

Tissues and cells were collected and lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors (NCM Biotechnology, Jiangsu, China) for 30 minutes at 4°C. The concentrations of proteins were measured using a BCA Protein Assay Kit (Beyotime Biotechnology). Equal amounts of proteins (30 µg) were separated by SDS-PAGE and transferred to polyvi-nylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% skim milk for 1 hour at room temperature, the membranes were incubated with primary antibodies at 4°C overnight. The membranes were then incubated with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, USA). The protein bands were detected using enhanced chemiluminescence reagent (NCM Biotechnology). Primary antibodies were anti-GAPDH (1:8,000; Proteintech, Chicago, IL, USA), anti-cyclin D1 (1:10,000; Abcam, Cambridge, UK), anti-HOXA13 (1:1,000; Abcam), anti-Erk1/2 (1:1,000; Cell Signaling Technology), anti-p-Erk1/2 (1:1,000; Cell Signaling Technology), anti-vimentin (1:1,000; Cell Signaling Technology), anti-E-cadherin (1:1,000; Cell Signaling Technology), and anti-N-cadherin (1:1,000; Cell Signaling Technology).