Anti vegf a

Protein levels were assessed on Pooled lesioned hemispheres (two of each sex) at 6 or 24 h after HI using western blot for VEGF (anti-VEGFA, Santa Cruz; sc152, 1/200), IL1-β (Santa Cruz; sc1251, 1/200) and HIF-1α (Novus Biochemicals, NB-100-479, 1/200) were investigated and were normalized on whole proteins detection since β-actin (Sigma-Aldrich, A5441, 1/1000) undergoes significant regulation under development.
A larger panel of 111 cytokines was examined by multiplex protein arrays (Mouse XL cytokine array, Proteome Profiler^TM, R&D systems) in separate pools under manufacturer instructions. Separate pools equilibrated in sex were prepared separately (details in Supplementary Material).

Total protein extracts were obtained after washing the hearts with PBS and adding 300 ml of RIPA modified lysis buffer (50 mM NaCl, 50 mM Tris–HCl (pH 7.40), 1% Triton X-100, 1 mM EDTA, 1 mM PMSF; 2.5 g/l Protease Inhibitor Cocktail (Sigma-Aldrich Co., St. Louis, MO, USA), 1 mM Na₃VO₄, 1 mM NaF), or washing the cultured cells and scraped off the dishes with 50 µl of the same buffer. Then, the tubes were kept on ice for 30 min with swirling, and the samples were centrifuged at 7,000 g at 4°C for 10 min. The supernatants were stored at −20°C. Protein concentrations were determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad, USA) and bovine serum albumin (Sigma-Aldrich Co., St. Louis, MO, USA) as a standard (36 (link)). For Western blot analysis, total proteins were boiled in Laemmli sample buffer, and equal amounts of protein (40–50 µg) were separated by 10–12% SDS-PAGE. The gels were blotted onto a Hybond-P membrane (GE Healthcare, Madrid, Spain) and incubated with the following antibodies: anti-NOS2, anti-NOS3, anti-Arginase I (Arg-I), anti-CD31, anti-VEGF-A, and anti-α-actin (Santa Cruz Biotechnology, CA, USA). The blots were revealed by enhanced chemiluminescence in an Image Quant 300 cabinet (GE Healthcare Biosciences, PA, USA) following the manufacturer’s instructions. Band intensity was analyzed using the NIH-ImageJ software (37 (link)).

Cells were harvested and lysed with RIPA buffer (Thermo Scientific Inc., Boston, MA, USA) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and a phosphatase inhibitor cocktail (Sigma-Aldrich). Total protein concentrations were quantified using an RC/DC protein assay reagent (Bio-Rad, Hercules, CA, USA). Protein extracts were separated using 6 and 10% SDS-PAGE and transferred onto PVDF membranes (PALL, Westborough, MA, USA). Membranes were incubated in a blocking solution (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA) for 30 min and incubated with anti-VEGF-A, anti-VEGFR-1, anti-VEGFR-2, anti-phospho FAK, anti-phospho ERK1/2, anti-phospho PI3K, anti-phospho AKT, anti-phospho JNK, anti-phospho p38 antibodies (Santa Cruz Biotech. Inc.) at 1:2000 dilution in a blocking solution overnight at 4 °C, and probed with peroxidase conjugated secondary antibodies at 1:5000 dilution. Protein bands were detected using enhanced chemiluminescent Western blotting detection reagent (Thermo Scientific Inc.)
Also, protein extracts were immunoprecipitated using a mouse anti-phospho-Tyr antibody (Santa Cruz Biotech. Inc.) and an ImmunoCruz™ IP/WB Optima kit (Santa Cruz Biotech. Inc.). Immunoprecipitated proteins were subjected to 6% SDS-PAGE and Western blotting using anti-VEGFR-1 and anti-VEGFR-2 antibodies (Santa Cruz Biotech. Inc.)

Immunohistochemistry (IHC) staining was performed for prostate tissue section at thickness of 10μm. First, paraffin was removed from slides through a deparaffinization process. To avoid endogenous peroxidase activity, slides were incubated with 3% of H₂O₂ solution in methanol at room temperature for 15 minutes and then washed with PBS for 3 times (5 minutes each). Slides were prerestrained with normal goat or rabbit serum for 1hr. In primary antibody reaction stage, slides were incubated carefully with anti-VEGFA, anti-TNF-alpha, anti-IL1β, anti-IL-6, and anti-COX2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in a 1:500 dilution for a night at 4°C and washed with PBS for 3 times, 5 minutes each. For second antibody reaction, the slides were incubated with biotinylated secondary antibodies (1:1000) at room temperature for 1h and washed with PBS three times, five minutes each. Finally, diaminobenzidine tetrahydrochloride substrate (DAB) staining with streptavidin-HRP was performed to visualize antigen-antibody reaction. For each section, three different random fields were examined at least ×100 magnification.

Cell or tissue lysates were prepared with lysis buffer [200 mM Tris-Cl (pH 7.5), 1.5 M NaCl, 20 mM EDTA, 10% Triton X-100, 10 mM Na₃VO₄·12 H₂O, and 1% protease inhibitor cocktail (Sigma-Aldrich St. Louis, MO, USA)] on ice. Protein concentrations of samples were determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies were as follows: anti-SerRS (1:1,000; made in the laboratory), anti-VEGFA (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β-actin (1:5,000; Santa Cruz Biotechnology), and anti-MTA2 (1:1,000, Santa Cruz Biotechnology). All antibodies recognized both human and mouse proteins. After SDS-PAGE and western blotting, the bands were detected by chemiluminescence using an ECL kit (Thermo Fisher Scientific).