Vegf a antibody

The proteins for western blotting from each sample were extracted and separated by PAGD and transferred to a PVDF membrane (Cell Signaling Technology, MA, USA) at 300 mA for 2 h. The membrane was initially incubated in blocking buffer (5% bovine serum albumin) for 2 h at room temperature then incubated overnight at 4°C with primary GAPDH (1:1000, Cell Signaling Technology, MA, USA) or VEGF-A antibody (1:1000, Abcam, Cambridge, MA, UK) followed by 2 h of incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:3000, Beyotime, China). Protein expression was visualized by chemiluminescence using an Alpha Imager scanner (Tecan, Thermo Fisher Scientific, USA).

The expression of LC3B-I/II and VEGF were analyzed by western blot as previously described [15 (link)]. REVCs were treated as mentioned above. Cells were lysed in the RIPA buffer (Beyotime, China), and protein concentration was measured by using a BCA Protein Assay kit (Beyotime, China). Totally 40 μg cell lysate was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 120 V. The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) at 200 mA for 90 min. The membranes were blocked with 5% non-fat dried milk in TBST for 1 h at room temperature and then incubated with LC3B antibody (1:1000, Cell Signaling, USA), VEGF-A antibody(1:1000, abcam, USA) and GADPH rabbit pAb (1:1000, Hangzhou Xianzhi biology co., LTD, China) primary antibodies for overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:50000, Boster, China) for 1 h at RT. Then the membranes were incubated with ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) and imaged using a ChemiDoc XRS imaging system (Bio-Rad, USA). The gray scale intensity of bands was analyzed using the QuantityOne software (Bio-Rad, USA) and the signals of LC3B I/II and VEGF were normalized to that of GAPDH.

To confirm miR-126 function in vivo, 1 × 10⁷ SGC-7901 cells stably transfected with lenti-miR-126, lenti-miR-NC or lenti-anti-miR-126 were injected subcutaneously into the right armpit of three groups of 18–26g male BALB/c nude mice (8 mice/group). At 42 days after inoculation, all mice were sacrificed. The tumor masses were excised, weighed, photographed and subjected to immunohistochemistry and Western blot, using CD34 antibody (Maxim Biotech, USA) and VEGF-A antibody (Abcam, USA). All animal experiments were performed in accordance with institutional guidelines and were approved by the animal care review board at the Nanchang University.

The western blotting instruction was described previously [8 (link)]. In brief, the proteins from each sample were extracted and transferred to a PVDF membrane (Cell Signaling Technology, MA, USA). The membrane was initially blocked in 5% bovine serum albumin for 2 h and then incubated with primary CD63 (1 : 2000, Abcam, Cambridge, MA, UK), CD81 (1 : 1000, Cell Signaling Technology, USA), α-tubulin (1 : 1000, Cell Signaling Technology, MA, USA), VEGF-A antibody (1 : 1000, Abcam, Cambridge, MA, UK), and β-catenin (1 : 1000, Cell Signaling Technology, USA) at 4°C overnight, followed incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1 : 3000, Beyotime, China). An Alpha Imager scanner (Tecan, Thermo Fisher Scientific, USA) was used to visualize the protein expression by chemiluminescence.

We collected the FFPE samples of 19 patients who received anti-PD-1 immunotherapy to obtain the protein expression values of the chosen four gene pairs in the CICAMS cohort. Expression levels of eight genes were determined via the IHC method using an anti-human C-C motif chemokine ligand 2 (CCL2, MCP1) antibody (Cat# 25542-1-AP, Proteintech, USA), an anti-human vascular endothelial growth factor A (VEGFA) antibody (Cat# ab52917, Abcam, USA), an anti-human cyclin dependent kinase 1 (CDK1) antibody (Cat# ab133327, Abcam, USA), an anti-human C-X-C motif chemokine ligand 9 (CXCL9, MIG) antibody (Cat# 22355-1-AP, Proteintech, USA), an anti-human major histocompatibility complex, class II, DO beta (HLA-DOB) antibody (Cat# NBP1-87469, NOVUS, USA), an anti-human LCK proto-oncogene, Src family tyrosine kinase (LCK) antibody (Cat# ab32149, Abcam, USA), an anti-human interleukin 12A (IL-12A) antibody (Cat# ab131039, Abcam, USA), and an anti-human T-box 21 (TBX21) antibody (Cat# ab150440, Abcam, USA). Importantly, all IHC slides were assessed based on the evaluation method of the previously published study (30 (link)–33 (link)). Representative staining images of eight genes from the BRGPI model are shown in Supplementary Figure S2.

Sections were stained with the angiogenesis makers including antivascular endothelial growth factor (VEGFA) antibody (1 : 200, Abcam, Cambridge Biomedical Campus, Cambridge, UK) and anti-von Willebrand factor (VWF) antibody (1 : 200, Abcam). A number of endometrial glands in each section were manually counted under 4× magnification. The number of VEGFA and VWF-positive blood vessel density were counted from 5 randomly selected images of each section under 20× magnification.

Western blot was performed according to the standard protocol. Briefly, proteins were segregated on SDS-polyacrylamide gels and transmitted to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with specific primary antibodies: RREB1 antibody (#NBP1-71884, Novus Biologicals, USA), Argonaute 2 antibody (#ab32381, Abcam, USA), MMP14 antibody (#ab51074, Abcam, USA), VEGF-A antibody (#ab1316, Abcam, USA), and GAPDH antibody (#2118, Cell Signaling Technology, USA) overnight at 4°C. After the membranes were incubated with secondary antibodies, they were subjected to immunoblot analysis using an enhanced chemiluminescence (ECL) immunoblotting kit according to the manufacturer’s protocol.