Enhanced chemiluminescence system

Synaptic proteins were assayed by western blot as described previously [2–4 (link)]. Briefly, aliquots of brain homogenates were mixed with equal volumes of Laemmi loading buffer and boiled prior to gel electrophoresis. Equal amounts of protein were loaded and separated using SDS-PAGE (4–20%; Bio-Rad, Hercules, CA, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were then rinsed in TBST buffer and incubated overnight in TBST solution containing the primary antibody of interest (goat anti-PSD-95 and rabbit anti-synapsin-1; Abcam, Cambridge, MA, USA). The next day, blots were incubated with the appropriate peroxidase-linked secondary antibody, followed by visualization of protein-antibody complexes using the enhanced chemiluminescence system (Millipore, Billerica, MA, USA), and digital images were developed using a Licor C-Digit blot scanner (LI-COR Biotechnology, Lincoln, NE, USA). Immunoreactive bands were compared densitometrically using the blot scanner’s software. Membranes were stripped off using a stripping buffer (Thermo Fisher Scientific, Rockford, IL, USA), and then incubated with β-tubulin antibody (mouse anti-βIII-tubulin; Sigma-Aldrich, St. Louis, MO, USA) used as the loading control.

For the Western blot assay, cells were harvested in ice-cold phosphate-buffered saline 48 hours after transfection and lysed on ice in cold-modified radioimmunoprecipitation buffer supplemented with protease inhibitors. The protein concentration was determined using a bicinchoninic acid protein assay kit. Equal amounts of protein were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gels were electroblotted onto nitrocellulose membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 2 hours with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20, and incubated at 4°C overnight with primary antibody, VEGFA and GAPDH (Cell Signaling Technology, Danvers, MA, USA). Detection was performed after peroxidase-conjugated secondary antibodies using an enhanced chemiluminescence system (Millipore).

Forty-eight hours after transfection with four groups, the SH-SY5Y cells were washed with cold PBS and lysed in RIPA Lysis Buffer (Beyotime, China). Protein concentrations were determined using a bicinchonininc acid (BCA) assay kit (Pierce, USA). On a 10% SDS-PAGE gel, the total protein was separated and transferred to a PVDF membrane (Millipore, USA). GAPDH on the same membrane was used as a loading control. The primary monoclonal antibodies were anti-EFNB2 (ab150411, Abcam, UK) or anti-GAPDH (TA-08, ZSGB-BIO, China) and anti-mouse (31430, Thermo, USA) or anti-rabbit IgG (31460, Thermo, USA) were used as the secondary antibodies to further detect the signal. The target proteins were determined on membranes by an enhanced chemiluminescence system (Millipore, USA). The intensities of the immunoblots were quantified with a bio imaging (Syngene, UK) coupled to a personal computer. Expression level was accomplished by obtaining the ratio of the band density of EFNB2 to that of GAPDH from the same sample.