Ab81321

Tissue and cell samples were lysed in RIPA buffer (Beyotime Biotechnology, China). An equal amount (50 μg) of protein in each group was loaded and separated on 8–12% SDS-PAGE, then transferred onto 0.4 mm PVDF membranes (EMD Millipore, Billerica, MA, USA). After blocking in 5% skim milk for 1 h (h), membranes were incubated at 4 °C overnight with primary antibodies against Sema3A (ab199475, Abcam), Sema3A (ab23393, Abcam) neuropilin-1 (ab81321, Abcam), plexin-A1 (ab92346, Abcam), cyclinD1 (ab16663, Abcam), PCNA (ab29, Abcam, RRID: AB_303394), PDGFRβ (ab32570, Abcam, RRID: AB_777165), p-PDGFRβ (3173, Cell Signalling, RRID: AB_2252179), p53 (2524, Cell Signalling, RRID: AB_331743) and β-actin (A01010, Abbkine, RRID: AB_2737288). Then, membranes were incubated with secondary antibodies for 1 h at room temperature. All protein bands were visualized using ECL solution on a BioSpectrum Imaging System (UVP, Upland, CA, USA). The intensities of the bands were analysed using the ImageJ software package (National Institutes of Health, Bethesda, MD, USA). For immunoprecipitation, an equal amount (500 μg) of protein in each group was incubated with specific antibodies at 4 °C overnight with gentle rotation. The complexes were precipitated with Protein A/G agarose beads and analysed by western blotting.

RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) was utilized to isolate total protein from tissue samples, cells, or tumor xenografts. The isolated total protein was quantified with the Bradford Kit (Beyotime Institute of Biotechnology). Protein samples containing equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in a 10% gel and transferred onto polyvinylidene difluoride membranes. Blocking with 5% nonfat milk was performed at room temperature for 2 h to prevent nonspecific binding of antibodies. The membranes were next probed with primary antibodies against NRP1 (cat. # ab81321; 1:1000 dilution; Abcam) or GAPDH (ab181603; 1:1000 dilution; Abcam), followed by incubation with a goat anti-rabbit IgG horseradish peroxidase-conjugated antibody (ab6721; 1:5000 dilution; Abcam) (secondary antibody) at room temperature for 1 h and visualization with an enhanced chemiluminescence detection system (GE Healthcare, Chicago, IL, USA).