Ab5473

The proteins were extracted from the tissue of the peri-infarct area of rats and treated HUVECs, then quantified by the BCA protein assay kit. The membranes were incubated with antibodies against S1R (1:1000, ab253192, Abcam), JAK2 (1:1000, ab108596, Abcam), phosphorylated JAK2 (1:1000, ab32101, Abcam), STAT3 (1:1000, ab68153, Abcam), phosphorylated STAT3 (1:1000, ab267373, Abcam), VEGF (1:1000, ab214424, Abcam), bcl2 (1:1000, ab182858, Abcam), bax (1:1000, ab32503, Abcam), cleaved caspase-3 (1:1000, ab2302, Abcam), TGF-β1 (1:1000, ab215715, Abcam), phosphorylated VEGFR2 (1:1000, ab5473, Abcam) and GAPDH (1:1000, GB15004, Servicebio).Then, these were incubated with HRP-conjugated secondary antibodies (1:3000, GB23303, GB23301, Servicebio). The western blot images were then analyzed and calculated using Image J software.

Microglia were labelled using a rabbit primary antibody targeting ionized calcium binding adapter molecule 1 (IBA‐1) (019‐19741) from Wako (Richmond, VA, USA). Antibodies against basigin (Rabbit polyclonal antibody used for Western blot, ab64616; Rat monoclonal antibody used for immunofluorescence double labelling, ab34016), PECAM (ab9498), IGF‐1 (ab9572), IGF‐1 receptor (ab16890), VEGF (ab51745) and VEGFR‐2 (ab5473) were purchased from Abcam (Cambridge, MA, USA). HIF‐1α (MAB 5382) was obtained from Millipore (Billerica, MA, USA). Antibodies against AKT (9272), P‐AKT (4060), ERK (4695), P‐ERK (4370) and the MEK1/2 inhibitors PD98059, PI3K inhibitor LY294002 were from Cell Signaling (Andover, MA, USA). Antibodies against β‐actin were from Santa Cruz (Santa Cruz, CA, USA). Recombinant human IGF‐1 (100‐11) was from ProteinTech (Rochy Hill, NJ, USA). Secondary antibodies including goat anti‐rabbit conjugated to AlexaFluor 594/CY3 or AlexaFluor 488/FITC were purchased from Beijing ComWin (Beijing, China). SYBR Premix Ex Taq II and Multiscript RT were purchased from TaKaRa (Dalian, China).

Paraffin-embedded tissue was baked and deparaffinized before antigen retrieval. After 1 h of blocking with normal donkey serum, tissues were incubated with two unconjugated primary antibodies, anti-CD31 (2 μg/ml) (Cat# sc-1506, Santa Cruz Biotechnology,1:100) and anti-phospho-vascular endothelial growth factor receptor-2 (p-VEGFR-2) (7.5 μg/ml) (Cat# ab5473, Abcam, 1:100) or anti-endothelial cell-specific molecule 1 (ESM 1) (10 μg/ml) (Cat# ab103590, Abcam, 1:100) and anti-Apelin (1.3 μg/ml) (Cat# 11497-1-AP, Life Technologies, 1:100) at 4 °C overnight. The corresponding fluorochrome-conjugated secondary antibodies were applied for 1 h at room temperature on the next day, and DAPI was used to counterstain the nuclei. Slides were mounted and visualized under a confocal scanning microscope (FBV-1000; Olympus Corporation, Center Valley, PA) [22 (link)].

ProteinExt^® Mammalian Total Protein Extraction Kit (TransGen Biotech, China) was applied to extract the protein in tumor and renal tissues based on the instructions, and anti-VEGFA (ab1316; provided by Abcam, USA), VEGFR2 (ab39256; provided by Abcam, USA), β-actin (ab179467; purchased from Abcam, USA), P-VEGFR2 (ab5473; provided by Abcam, USA), RelA (ab32536; Abcam, USA) and c-mip (12851-1-AP; proteintech, China) primary antibodies as well as mouse anti-rabbit secondary antibodies (#7076, #7074; purchased from Cell Signaling Technology, USA) were used for Western blot. Gel-Pro Analyzer (provided by Media Cybernetics, MD, USA; Version 4.0) was used for protein analysis.

Cellular proteins were extracted with RIPA lysis buffer (Sigma Aldrich), separated using SDS-PAGE (Beyotime), and transferred to polyvinylidene fluoride membranes (Beyotime). Membranes were then blocked in 5% skim milk and treated with anti-E2F7 (1 : 1000, ab56022; Abcam, UK), VEGFR2 (1 : 300, bs-10412R; Bioss, China), p-VEGFR2 (1 : 1000, ab5473; Abcam, UK), GAPDH (1 : 3000, bs-2188R; Bioss, China), and mouse anti-rabbit secondary antibody (1 : 5000, bs-0295M-HRP; Bioss, China). Western blots were visualized using the ECL system (Beyotime).