Sc 133086

Western blot analysis was conducted using an SDS-PAGE electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, the total protein content was extracted from MCF-7, MCF-10A and MDA-MB-231 cell lines using a RIPA buffer (P0013C, Beyotime, Shanghai, China). Protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes (88,520, Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently blocked 12 h at 4 °C with 5% skimmed milk containing TBST (Tris-buffered salt solution, containing 50 mmol/L Tris–HCl, 150 mmol/L NaCl, 0.1% v/ v Tween-20, pH 7.4) solution. Antibodies against ALB (ab106582, Abcam, San Francisco, CA, USA), ANGPTL7 (orb350273, Biorbyt, Cambridge, UK), BLOC1S5-TXNDC5 (abx230909, Abbexa, Ltd, Cambridge, UK), IL6 (orb10911, Biorbyt, Cambridge, UK.), and NGB (sc-133086, Santa Cruz, Dallas, TX, USA.), and GAPDH (ab8245, Abcam, San Francisco, CA, USA.) were used as primary antibodies. The samples were incubated with horseradish peroxidase–conjugated secondary antibodies at 37 °C for 1 h. The membrane was imaged using an Amersham Imager 600 (GE Healthcare UK Limited, Little Chalfont, UK).

HCT116 cells were seeded on a microcover slip and then transfected with pCMV6-Entry-NGB plasmids. After 48 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and then blocked with blocking buffer. Afterward, slides were incubated with primary antibody at 4 °C. After 20 h, the cells were incubated with Alexa Fluor 594- or 488-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (ab150113, ab150080, Abcam) for 1 h at room temperature in the dark. All slides were next counterstained with 4'-6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology). Photomicrographs were captured with a confocal laser scanning microscope. All assays were performed three times. Primary antibodies included anti-NGB (sc-133086, Santa Cruz Biotechnology), anti-GPR35 (TA313953, Origene technologies), anti-Myc tag (#2278; Cell Signaling Technology), anti-CD31 (#3528, Cell Signaling Technology), and anti-VEGFR2 (sc-6251, Santa Cruz Biotechnology).