Vegfr2

Scutellarin (purity 98%) was purchased from Jiexiang Pharmaceutical Industry Ltd. (Sichuan, China). Chitosan (deacetylation degree of 90% and average molecular weight of 450 kDa) was purchased from Shanghai Bo’ao Biological Technology Co, Ltd (Shanghai, China). Deoxycholic acid was purchased from Acros Organics Corp (Antwerp, Belgium). N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) was obtained from Shanghai Medpep Co, Ltd (Shanghai, China, AR). FITC was sourced from Sigma-Aldrich (St Louis, MO). Vitamin B12 was purchased from Sigma (St Louis, USA). CDI and dimethyl sulfoxide (DMSO) were acquired from Aladdin Reagent Company (Shanghai, China). Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies Inc, (Kumamoto, Japan). Zebra fish embryos were kindly provided by the Zebra fish Model Animal Facility at the Institute of Clinical and Translational Research of Sun Yat-sen University. Primary antibodies against VEGF, VEGFR2, vWF and Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Tissue or cells were lysed in 2x Laemmli buffer and boiled for 5 min at 100 °C followed by centrifugation at 20,000 × g for 15 min at 4 °C. The protein extracts were subjected to standard Western blot analysis which was performed. The following antibodies were used for western blotting: Rabbit polyclonal antibody against CCM3 was generated (Invitrogen) against full-length recombinant human CCM3 protein expressed and purified from Escherichia coli. β-actin (mouse, A1978) and β-actin (mouse, A5441) were from Sigma; p-Caveolin-1 (rabbit, 611339) was from BD Pharmingen; Abl (rabbit, 2862s), p-Akt (rabbit, 9271), Akt (rabbit, 9272), p-MLC2 (rabbit, 3674), PDGFR-β (rabbit, 3168), p-Tie2 (rabbit, 4221), p-Tie2 (rabbit, 4226), Tie2 (rabbit, 4224), and VEGFR2 (rabbit, 2479) were from Cell Signaling Technology. Caveolin-1 (rabbit, sc894) was from Santa Cruz. Angpt2 (rabbit, ab8452), p-Abl (rabbit, ab4717), and N-Cadherin (rabbit, ab76057) were from Abcam; Angpt2 (AF7186) was from R&D Systems. All primary antibodies were diluted 1:1000. For data presented in the same figure panel, the samples were derived from the same experiment and that gels/blots were processed in parallel. Uncropped blots and gel images were provided in the Source data file.

Serum-starved subconfluent LEC cultures were incubated in the presence or absence of AD0157 for 2 h and then challenged for 30 additional minutes with rhVEGF-C (400 ng/mL), rhVEGF-C156S (500 ng/mL; R&D Systems), or rhVEGF-A (100 ng/mL; R&D Systems). Cell lysates were analyzed by SDS-PAGE, electrotransferred to nitrocellulose membranes (GE Healthcare Life Sciences), and blots were probed with primary antibodies against pVEGFR-3 (1/1000; Cell Application), VEGFR-3 (1/1000; Millipore), pVEGFR-2, VEGFR-2, pERK1/2, ERK1/2, pAkt, Akt, (1/1000; Cell Signaling), and GADPH (1/2000; Millipore). Detection was carried out with chemiluminescence system ECL Western Blotting Substrate (Pierce), and bands were quantified and expressed as phosphorylated protein/total protein ratio.

Western blotting was performed as previously described [16 (link)]. Briefly, cell lysates were prepared from parental wild type or miR-214KO PC3 and MDA-PCa-2b cells using lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Cell lysates (40 μg) were separated and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4 °C with anti-PCNA, VEGFR2, E-Cadherin, N-Cadherin, Vimentin, PTK6, and GAPDH (Cell Signaling Technology), CD31 (Life Technologies), VEGFA (Abcam), CXCR4, SESN3, PD-L1, ALK, and SAA1 (Abclonal Technology, and the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. All primary antibodies were used at a concentration of 1:1000. Following the incubation, membranes were washed, and protein bands were detected with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

The methods were performed as previously published [19 (link)]. Human NB cells SK-N-AS were cultured in 6-well plates at the density of 2 × 10⁶ cells/well. The NB cells were treated with SSA (0, 7.5, 10, 12.5, 15, or 17.5 μM) for 24 h. After this, the NB cells were added with the RIPA buffer for cell lysing. 8,000 × g centrifugation for 15 min at 4̊C was used to collect the total protein. The total protein was determined by bicinchoninic acid assay (Beyotime, Shanghai, China). Then, 50 μg of the protein was separated by 10% SDS-PAGE. The protein was then transferred to the PVDF membranes. Western blotting was performed as previously published [23 (link)]. All of the primary antibodies used were rabbit monoclonal antibodies (mAb). The mAbs of cleaved PARP, PARP, cleaved caspases (7, 9), caspases (7, 9), Bcl-2, Bax, E-cadherin, N-cadherin, Vimentin, Slug, Snail, ZO-1, p-Akt, Akt, p-Src, Src, p-VEGFR2, VEGFR2, and β-actin were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The bands of the proteins were visualized by ECL (Applygen Technologies Inc., Beijing, China) with a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai, China).

The intestinal samples were processed as previously reported. The total protein concentration was quantified using the Bradford protein assay. The different samples (30 μg protein) were first subjected to electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes for primary antibody incubation. The primary antibodies utilized in the current research included p-eNOSSer1177 (Cell Signaling Technology, USA), eNOS (Cell Signaling Technology), CD31 (Cell Signaling Technology), VEGFR2 (Cell Signaling Technology) and GAPDH (Santa Cruz Biotechnology). The appropriate secondary antibodies were incubated to visualize the immunoreactive bands. β-actin (Sigma) was used as a loading control. The relative intensities of the target bands were measured utilizing Kodak 1D 3.5.4 software (Kodak Scientific Imaging System, Rockville, MD, USA).

KHOS and U2OS cells were obtained from American Type Culture Collection (ATCC), and we used short tandem repeat (STR) analysis for the authentication of the two cells at Beijing Microread Genetics Co., Ltd. Cells were cultured in RPMI 1640 medium (HyClone) supplemented with 1% penicillin and streptomycin (Invitrogen) and 10% fetal bovine serum (Gibco) in an environment with a temperature of 37°C and 5% CO2.
The antibodies we used in relevant experiments were as follows: antibodies against p-STAT3, STAT3, p-cofilin, cofilin and VEGFR2, which we purchased from Cell Signaling Technology in United States; anti-PDL2, p-LIMK2, LIMK2 and the RhoA activation assay, which we obtained from Abcam in United States; and anti-GAPDH, which we purchased from Santa Cruz Biotechnology. Y-27632 2HCl was purchased from Selleck.