Anti phosphotyrosine antibody

Western immunoblotting analysis was performed to examine tyrosine phosphorylation in the sperm after incubation in coculture medium. Twenty micrograms of sperm protein were separated on 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and then transferred to a 0.45-mm hybond polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Germany). The membranes were blocked with 3% bovine serum albumin (BSA) for 1 h at 25°C. The membrane was subsequently incubated with the primary antibody (anti-phosphotyrosine antibody; Santa Cruz, USA; 1:500) overnight at 4°C. The membrane was washed with 1× tris-buffered saline with 0.1% Tween® 20 detergent (TBST) for 5 min 3 times and then incubated with a secondary antibody (goat anti-mouse immunoglobin G-horseradish peroxidase (IgG HRP) conjugated; Santa Cruz, USA; 1:1000) for 2 h at room temperature. The membrane was washed 3 times with 1× TBST for 5 min and visualized using an ECL plus Western blot detection system (Thermo Fisher Scientific Inc., Hampton, USA). The antigen-antibody reaction was detected using a Luminescent Image Analyzer (Imagequant LAS 4000, Sweden).

DC protein assay (Biorad, Hercules, CA, USA) was performed to the samples after the experimental incubations in order to quantify the total protein amount. Samples were then solubilized in Laemmli buffer with 2% betamercaptoethanol and heated at 95 °C (5 min). Solubilized protein samples were loaded (30 μg) and run in SDS-PAGE. Next, the proteins were transferred to nitrocellulose membranes by western blot analysis. Membranes were blocked in PBS/Tween. 1:2000 Anti-phosphotyrosine antibody (Santa Cruz, CA, USA) was used. Secondary rabbit antibody conjugated to infrared fluorescent dyes excitable at 800 nm (IRDye 800CW, Licor, Lincoln, NE, USA) was incubated at 1:25,000 for 1 h and then washed. In order to visualize the antigens, 800 nm laser scanner (Odyssey, Licor, Lincoln, NE, USA) was used. Quantitative analysis of proteins on the membranes was performed by ImageJ (USA).

Cell lysates were electrophoresed on SDS-polyacrylamide gels and transferred onto nitrocellulose blotting membranes (GE Healthcare, Freiburg, Germany). Blots were probed with monoclonal anti-Smad1, anti-Smad2/3, anti-phospho-Smad1/5, or anti-phospho-Smad2/3 antibodies (Cell Signaling Technologies, Danvers, MA, USA), followed by a horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich). Signals were detected using a chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK) and a luminescent image analyzer (LAS-3000; Fujifilm, Tokyo, Japan).
To evaluate VEGF receptor phosphorylation, HUVEC extracts (400 μg of protein) were mixed with 4 μg of VEGFR1 or VEGFR2 polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Protein G-agarose beads (Invitrogen/ThermoFisher Scientific), and incubated at 4°C overnight with gentle rotation. Immune complexes were precipitated by centrifugation and analyzed by western blotting using an anti-phospho-tyrosine antibody (Santa Cruz Biotechnology).