Anti vegfa

Western blot was performed as previously described (35 (link)). Primary antibodies used were: anti-TNFAIP6 (1:1000, Proteintech, China), anti-TLR6 (1:1000, ABclonal, China), anti-FAS (1:3000, Proteintech, China), anti-CCL3 (1:1000, Proteintech, China), anti-ICAM-1 (1:3000, Proteintech, China), anti-CCL2 (1:3000, Proteintech, China), anti-CXCR4 (1:3000, Proteintech, China), anti-VEGFA (1:2000, Proteintech, China), anti-β-Tubulin (1:20000, Proteintech, China) and anti-GAPDH (1:2000, Servicebio, China).

The glioma cells were lysed using RIPA buffer with Protease and Phosphatase inhibitor (Beyotime). The total protein concentration was examined using a BCA kit. Equal amounts of total protein (30 μg) were electrophoresed in 10%–12% SDS‐PAGE and electro‐transferred onto NC membrane (GE). After blocking with 5% non‐fat dry milk in TBST for 1 h at room temperature (RT), the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with HRP‐labeled secondary antibodies (Proteintech) for 1 h at RT. An enhanced chemiluminescence reagent (Millipore) was used to detect protein expression value. The relative quantity of proteins was analyzed using the Image J software. The primary antibodies used are as follows: anti‐EZH2 (1:2000, #66476‐1‐Ig, Proteintech), anti‐VEGFA (1:1000, #66828‐1‐Ig, Proteintech), anti‐VEGFR2 (1:200, #sc‐6251, Santa‐Cruz Technology), anti‐Phospho^Tyr1175‐VEGFR2 (1:1000, #2478, CST), anti‐AKT (1:1000, #9272, CST), anti‐ Phospho^Thr308‐AKT (1:1000, #13038, CST), anti‐ERK1/2 (1:1000, #4695, CST), anti‐Phospho^{Thr202/Tyr204}‐ERK1/2 (1:1000, #4370, CST), anti‐GAPDH (1:5000, #60004‐1‐Ig, Proteintech), and anti‐β‐tubulin (1:5000, #10094‐1‐AP, Proteintech).

The cells were fixed in 4% paraformaldehyde (PFA) in 1× PBS at room temperature for 20 minutes. The cells were first pre‐treated and then blocked in 1× PBS with 0.5% Triton X‐100 and 5% BSA for 1 hour. Next, the samples were incubated with anti‐VEGFA (1:200 dilutions, ProteinTech) overnight at 4°C, followed by incubation with secondary antibodies goat anti‐Rabbit Alexa 488 (1:200 dilutions, ZSGB‐BIO). The samples were then analysed with a laser confocal microscope (OLYMPUS IX83, UltraVIEW VoX) with a 640 nm pulse. The images were collected with the Volivity software containing Acquisition, Quantitation and Visualization modules.

All cells were lysed in RIPA-Buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease and phosphatase inhibitors (Thermo Scientific, USA) on ice for 30 min, followed by centrifugation at 12,000 g for 15 min. Protein concentrations were calculated using the Pierce BCA protein assay kit (Thermo Scientific, USA). Equivalent amounts of protein samples (25 µg/lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline and 0.1% Tween 20 buffer and then incubated with primary antibody overnight at 4°C. Primary antibody including anti-VEGFA (Proteintech, MA5-13182), anti-ABCC1 (Abcam, ab91451), anti-MMP2 (Abcam, ab97779), anti-MMP9 (Abcam, ab38898), anti-Vimentin (Abcam, ab28151), anti-SNAIL (Cell Signaling Technology, #3879), GAPDH (Cell Signaling Technology, #5174), β-actin (ab179467). Membranes were incubated with goat anti-rabbit or anti-mouse HRP-conjugated secondary antibody (Beyotime Biotechnology, Shanghai, China) for 1 h at room temperature. Signal detection was performed using the ultrasensitive ECL plus Detection Reagent (Millipore, USA) and a ChemiDoc Touch Imaging System. The intensity of the bands was analyzed using the Quantity One software (Bio‐Rad, Hercules, CA, USA).

Antibodies and agents used in this study were as follows: anti-PDIA4 (Proteintech, Cat. No. #14,712–1-AP), anti-ATF6 (Proteintech, Cat. No. #24,169–1-AP), recombinant anti-XBP1 antibody (Abcam, Cat. No. #ab220783), anti-VEGFA (Proteintech, Cat. No. #19,003–1-AP), anti-Flag (Proteintech, Cat. No. 20543–1-AP), anti-HA (Proteintech, Cat. No. #66,006–2-Ig), anti-GAPDH (Proteintech, Cat. No. #10,494–1-AP), anti-HIF-1α (Proteintech, Cat. No. #20,960–1-AP), anti-β-Actin (Proteintech, Cat. No. #81,115–1-RR), anti-CD31 (Proteintech, Cat. No. 311265–1-AP), tunicamycin (Cat. No. #HY-A0098, MedChemExpress), temozolomide (Cat. No. HY-17364, MedChemExpress), human-VEGFA (Cat. No. HZ-1038, human VEGF-165 recombinant protein, Proteintech), and bevacizumab (Cat. No. #HY-P9906, MedChemExpress).

Protein in HUVECs was extracted with RIPA buffer containing PMSF (Solarbio, Beijing, China). The supernatant of the lysis and exosome derived from HCT116 and SW480 was quantified with a BCA kit. Then, more details are provided below [11 (link)]. The following antibodies were used: anti-CD9 (Abcam, ab263019); anti-CD63 (Abcam, ab134045); anti-TSG101 (Abcam, ab125011); anti-CD34 (Proteintech, 14,486–1-AP); anti-Integrin β1 (Proteintech, 12,594–1-AP); anti-VEGFA (Proteintech,19,003–1-AP); anti-PDK2 (Abcepta, AP7039b); anti-Akt (Abcam, ab179463); anti-p-Akt (Ser473); anti-GAPDH (Proteintech, 60,004–1-Ig). IF and IHC was performed as previously described [11 (link)]. IF was performed using anti-CD34 (Proteintech, 14,486–1-AP). Then, the image of IF was obtained using the LSM880 confocal microscope system (Zeiss, Jena, Germany). IHC was performed using anti-CD31 (Proteintech, 11,265–1-AP) and anti-CD34 (Proteintech, 14,486–1-AP). The images of IHC were acquired using a fluorescence microscope system (Olympus, Tokyo, Japan).