Yt5845

Cell immunofluorescence staining was conducted to evaluate the expression of proteins related to angiogenesis and cell apoptosis. HUVECs were plated in 6‐well plates (Corning, USA) at a density of 9.6 × 10⁵ cells and treated as mentioned above. Then, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min, permeabilized and blocked with 0.5% Triton X‐100 (Biofroxx, Germany) and 1% BSA (Biosharp, Anhui, China) for 20 min. Then, the samples were incubated together with the following diluted vascular endothelial growth factor 2 (VEGFR2) (1:200, YT5845, Immunoway) and S6K (1:200, YT3555, Immunoway) primary antibody at 4°C overnight. Next, they were cultured with secondary antibody anti‐rabbit IgG [1:1000, 4413, Cell Signalling TECHNOLOGY(CST)] under ambient temperature for 2 h. Then, the cell nucleus was stained with Hoechst 33342 (Solarbio, Beijing, China), and the cytoskeleton was stained with phalloidin (Solarbio, Beijing, China). Samples were fixed with anti‐fluorescence quenching sealed tablets.

Ischemic brain tissue (cell protein) extract was assayed with a BCA kit according to the manufacturer’s protocols towards obtaining accurate concentration of proteins and then added with 1/4 volume of protein buffer and denatured by heating. Aliquots of the samples (40 μg) were analysed through SDS-PAGE gel. Following this, the gel was transferred and then blocked and incubated with different corresponding primary antibodies against HIF-1α (ab197493, Abcam, Danvers, MA), VEGF (A12303, ABclonal, Wuhan, China), VEGFR2 (YT5845, Immunoway, Plano, TX), PLCγ1 (AF8390, Affinity, San Francisco, CA), PKC (YT3752, Immunoway, Plano, TX), eNOS (CPA9156, Bejing, China) overnight. Subsequently, the target membranes were incubated with a secondary antibody. Proteins were detected using the ECL kit and the results were analysed using Image Lab analysis software (Bio-Rad Laboratories, Inc., Hercules, CA).