Ab83042

Protein samples were extracted by RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was measured using Bicinchoninic Acid protein assay kit (Sigma-Aldrich; Merck KGaA). Next, 25 µg protein was separated by 10% SDS-PAGE and transferred to PVDF membranes. And the protein was blocked at room temperature for 2 h with 5% non-fat milk. Then, protein samples were incubated with FZD4 (ab83042, 1:1,000 dilution, Abcam, rabbit polyclonal antibodies) and GAPDH (ab8245, 1:1,000 dilution, Abcam, mouse monoclonal antibody) primary antibodies overnight at 4°C. After washing with TBST, protein samples were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (ab205719, 1:5,000 dilution; Abcam). Protein bands were visualized by ECL kit (Beyotime Institute of Biotechnology) and were quantified with Image Lab Software (Bio-Rad Laboratories, Inc.).

Total protein from corneas was extracted on ice with RIPA lysis buffer in the presence of freshly added protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). A total of 10 μg/lane protein extract was loaded onto a 4%–20% gradient SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Bio-Rad Laboratories). Nonspecific binding was blocked with 5% nonfat milk or 5% BSA in Tris-buffered saline with Tween20 (TBST) as recommended for each antibody. The membrane was incubated with rabbit anti-VEGF (ab46154; Abcam, Cambridge, MA, USA), anti-Angpt1 (ab95230; Abcam), anti-Tie2 (catalog [Cat.] no. 7403; Cell Signaling, Danvers, MA, USA), anti-phospho-Tie2 (AF2720-SP; R&D Systems, Minneapolis, MN, USA), anti-PI3K (p85) (Cat. no. 4292; Cell Signaling), anti-phospho-PI3K (p85) (Cat. no. 4228; Cell Signaling), anti-Akt (ab8805; Abcam), anti-phospho-Akt1 (ab81283; Abcam), anti-Fzd4 (ab83042; Abcam), anti-LRP6 (Cat. no. 3395S; Cell Signaling), anti-phospho-LRP6 (Cat. no. 2568S; Cell Signaling), anti-N-p-β-catenin (Cat. no. 4270; Cell Signaling), or anti-β-catenin (Cat. no. 8480S; Cell Signaling) antibodies overnight at 4°C. IRDye 800CW goat anti-rabbit IgG (Cat. no. 926-32211; LI-COR, Lincoln, NE, USA) was used as the secondary antibody, and mouse anti-GAPDH antibody (ab8245; Abcam) was used as an internal standard.

Total protein was extracted by cell lysate (BB-3209, Bestbio, Shanghai, China), separated by SDS-PAGE, and then transferred onto a polyvinylidene fluoride membrane. After the membrane had been blocked for 1 h, it was incubated with primary antibodies of rabbit polyclonal antibodies to FZD4 (1 μg/mL, ab83042), β-catenin (1:4,000, ab6302), c-myc (1:500, ab39688), cyclinD1 (1:500, ab61758), E-cadherin (1:500, ab15148), Vimentin (1:1,000, ab137321), Snail (1:500, ab82846), Slug (1:100, ab75629), p-GSK-3β^Ser9 (1:500, ab131097), and mouse monoclonal antibody to GSK-3β (1:500, ab93926) overnight at 4°C (all antibodies above were purchased from Abcam, Cambridge, MA, USA). The membrane was then incubated with the secondary antibody goat anti-rabbit antibody to immunoglobulin G (IgG) (A21020, Abbkine, USA, 1:1,000) for incubation for 1 h at 37°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was regarded as the loading control. The expression of target protein is equal to the gray value of target protein bands divided by the gray value of GAPDH.