Anti sfrp1

Total cell protein extraction was performed by using lysis buffer (Invitrogen, Carlsbad, CA, USA). For Western blotting, samples were denatured for 5 min at 95°C, separated on a 4–12% polyacrylamid gel and then transferred to a nitrocellulose membrane (room temperature, 1 h, Bio-Rad). Membranes were blocked with 5% skim milk in TBS-T for 1 h at room temperature and then incubated with the first antibody overnight at 4°C. The following primary antibodies were used: anti-SFRP1 (Santa Cruz, sc-13939, CA, USA), anti-BDNF (Santa Cruz, sc-546, CA, USA), anti-LY96 (Abcam, ab24182, Cambridge, UK) and anti-β-actin (Sigma-Aldrich, A5316, Deisenheim, Germany). Afterwards membranes were washed three times with TBS-T and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (DAKO, Glastrup, Denmark). After three washes with TBS-T, the signal was detected by chemiluminescence (Pierce ECL, Thermo Scientific, Rockford, IL, USA).

Total protein was extracted. With the addition of 10% SDS/PAGE, 20 μg protein samples were added into each well. Next, samples were added with primary antibody rabbit polyclonal anti-SFRP1, vascular endothelial growth factor (VEGF), c-Myc, cyclin D1 and β-catenin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, U.S.A.), and incubated at 4°C overnight. The next day, samples were incubated with secondary antibody POD–conjugated goat anti-rabbit antibody (1:5000) at room temperature for 30 min and then added with horseradish peroxidase (HRP, Bio-Rad Laboratories, Hercules, CA, U.S.A.) for developing. The intensity of protein bands was analyzed by Image Quant 350 and Image Quant TL-1 (GE Healthcare, Fairfield, CT, U.S.A.), with β-actin as the internal reference.

Western blot analysis was performed using anti–ROCK-2 (1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti–β-catenin (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-RUNX2 (1:500; Abcam), anti–p-SAPK/JNK (1:1000; Cell Signaling Technology), anti-SAPK/JNK (1:1000; Cell Signaling Technology), anti-SMA (1:500; Santa Cruz), anti-Wnt5a (1:1000; Abcam), anti-Wnt3a (1000; Millipore), anti-sFRP5 (1:2000; Thermo Fisher), anti-sFRP4 (1:500; Santa Cruz), anti-sFRP3 (1:500; Santa Cruz), anti-sFRP2 (1:500; Santa Cruz), anti-sFRP1 (1:500; Santa Cruz), and anti-GAPDH (1:5000; Flarebio, College Park, MD, USA) antibodies. Blots were immunolabeled using horseradish peroxidase-conjugated secondary antibody and incubated with chemiluminescent substrate (Pierce ECL Plus Western Blotting Substrate; Thermo). The Western blots were quantified using ImageJ software (National Institutes of Health).