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.
P vegfr2
Manufactured by Cell Signaling Technology
Sourced in United States, Germany, China
P-VEGFR2 is a phospho-specific antibody that targets the phosphorylated form of the vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 is a receptor tyrosine kinase that plays a crucial role in angiogenesis and vascular permeability. The phosphorylation of VEGFR2 is a key step in the activation of this signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Vegfr2, by Cell Signaling Technology (22 mentions)
P akt, by Cell Signaling Technology (14 mentions)
P erk1 2, by Cell Signaling Technology (6 mentions)
P stat3, by Cell Signaling Technology (6 mentions)
Stat3, by Cell Signaling Technology (6 mentions)
P pdgfrβ, by Cell Signaling Technology (5 mentions)
β actin, by Cell Signaling Technology (5 mentions)
P erk, by Cell Signaling Technology (5 mentions)
Erk1 2, by Cell Signaling Technology (4 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (4 mentions)
Bcl 2, by Cell Signaling Technology (4 mentions)
P src, by Cell Signaling Technology (3 mentions)
P egfr, by Cell Signaling Technology (3 mentions)
Pdgfrβ, by Cell Signaling Technology (3 mentions)
Survivin, by Cell Signaling Technology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Vegfr1, by Cell Signaling Technology (2 mentions)
38 protocols using p vegfr2
1
Modulation of VEGFR Signaling by AD0157
2
Molecular Mechanisms of Fibrosis Regulation
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Antibodies to p‐PDGFR‐β, p‐VEGFR2, p‐Src, Src, p‐STAT3, STAT3, p‐Smad3, Smad3, TGF‐β1, p‐NF‐κBp65, p‐Akt, Akt and β‐Actin were purchased from Cell Signaling Technology. TGF‐β1 and antibodies to type I collagen and fibronectin were purchased from Santa Cruz Biotechnology. p‐FGFR1 antibody was purchased from Life Span Biosciences. NF‐κBp65 antibody was purchased from Prosci Inc. Antibodies to CD68, CD31, MMP‐2, TIMP‐2, E‐cadherin, vimentin, Snail, Twist, and MCP‐1, TNF‐α, IL‐1β and IL‐6 ELISA assay kits were purchased from Abcam Inc. Nintedanib was purchased from Cayman. α‐SMA antibody, CG, Cell Counting Kit‐8 (CCK‐8) proliferation assay kit and all other chemicals were purchased from Sigma.
3
Neuroblastoma Cell Apoptosis and EMT Assay
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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).
4
Lung Cancer Cell Lines for Therapeutic Evaluation
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The following cell lines were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences: Calu-1 (human squamous cell carcinoma), NCI-H358 (human lung adenocarcinoma), NCI-H460 (human lung large-cell carcinoma), NCI-H1975 (human lung adenocarcinoma), NCI-H1650 (human lung adenocarcinoma), 95D (human giant cell lung carcinoma cell lines with high metastatic potential), NCI-H292 (human lung mucoepidermoid carcinoma-lymph node metastatic strain), A549 (human lung adenocarcinoma), and NCI-H1299 (human lung adenocarcinoma-lymph node metastatic strain). McCoy’s 5A and RPMI-1640 media were from Sigma Aldrich Co. (St Louis, MO, USA), FBS was from Si Ji Qing (Hangzhou, China). Apatinib (selective VEGFR2 inhibitor) and S3I-201 (inhibitor of STAT3) were purchased from Selleckchem.com (Houston, TX, USA). VEGFR2, HIF-1α, Akt, ERK1/2, p-38, p-VEGFR2, p-Akt, p-ERK1/2, p-p38, STAT3, and p-STAT3 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Apoptosis and cell cycle kits were from Nanjing Keygentec (Nanjing, China).
5
Molecular Profiling of Angiogenic Signaling Pathways
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VEGF-A165 was obtained from Sigma-Aldrich (St. Louis, MO; V5765). SiRNA for FUNDC1, IP3R1, VEGFR2, serum response factor (SRF), and controls were obtained from Santa Cruz Biotechnology (sc-36563, sc-91118, sc-42475, and sc-29318; Dallas, Texas). Antibodies against the following proteins were used as the primary antibodies: CD31 (BD Pharmingen, San Jose, CA; 550274; Cell Signaling Technology, Danvers, MA;77699); β-actin and GAPDH (Santa Cruz Biotechnology, Dallas, Texas; sc-47778 and sc-137179); PCNA, Calreticulin, Cyt C, VDAC1, mitofusin-2 (MFN2), SRF, phosphorylated (p) SRF, VEGFR1, VEGFR2, pVEGFR2, and VEGFR3 (Cell Signaling Technology, Danvers, MA; 13110, 12238, 4280,4661, 9482, 5147,4261, 2893, 2478, 2479, 3408); IP3R1 (Abcam, Cambridge, MA; ab5804; ThermoFisher, Waltham, MA, PA1-901); calnexin (Abcam, Cambridge, MA; ab22595); FUNDC1 (Aviva Systems Biology, San Diego, CA; ARP53281), FUNDC1 (Novus Biologicals, LLC, Littleton CO, NBP1-81063; EMD Millipore Corporation, Temecula, CA, ABC506).
6
Evaluation of Gefitinib and Sunitinib on HUVEC and Lung Cancer Cells
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Human umbilical vein endothelial cells (HUVECs) were purchased from Sciencell (CA, USA), and human lung cancer cell lines, HCC827 and H3255, were purchased from American Type Culture Collection (VA, USA). HUVECs were cultured in endothelial cell medium (ECM) kit (Sciencell) containing 5% fetal bovine serum (FBS) and 1% endothelial cell growth supplement. The human lung cancer cell lines were cultured in medium 1640 (Thermo Fisher Scientific, MA, USA) containing 10% FBS (Thermo Fisher Scientific), and 100 U/mL penicillin and 0.1 mg/mL streptomycin were added to the medium. All cells were cultured in an incubator at 37°C with 5% CO2.
Gefitinib and sunitinib were purchased from Selleck (TX, USA). CM082 was provided by Betta Pharmaceutica Co., Ltd. (Hangzhou, China). Recombinant human VEGF was purchased from R&D Systems (MN, USA). Antibodies for VEGFR2, p‐VEGFR2, ERK, p‐ERK, AKT, p‐AKT, p‐STAT3, and STAT3 were purchased from Cell Signaling Technology (MA, USA). Antibodies for VEGF‐A, CD31, Ki‐67, β‐actin, and β‐tubulin, and horseradish peroxidase‐conjugated antirabbit/mouse IgG antibodies, were purchased from ABclonal Technology (Wuhan, China).
Gefitinib and sunitinib were purchased from Selleck (TX, USA). CM082 was provided by Betta Pharmaceutica Co., Ltd. (Hangzhou, China). Recombinant human VEGF was purchased from R&D Systems (MN, USA). Antibodies for VEGFR2, p‐VEGFR2, ERK, p‐ERK, AKT, p‐AKT, p‐STAT3, and STAT3 were purchased from Cell Signaling Technology (MA, USA). Antibodies for VEGF‐A, CD31, Ki‐67, β‐actin, and β‐tubulin, and horseradish peroxidase‐conjugated antirabbit/mouse IgG antibodies, were purchased from ABclonal Technology (Wuhan, China).
7
Immunohistochemical Analysis of Xenograft Tissue
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Samples for these experiments were obtained from the xenograft experiment and fixed in 10% paraformaldehyde, embedded in paraffin, and sectioned. The tissue sections were then subjected to immunohistochemical staining with the Novolink Polymer Detection System (Leica Biosystems). The sections were stained for p-VEGFR2 (Tyr951, Cell Signaling Technology), Ki-67 and p-FAK (Tyr397) (Santa Cruz Biotechnology), and the nuclei were counterstained with hematoxylin.
8
Molecular Mechanisms of Anti-VEGF Therapies
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Vascular endothelial growth factor was purchased from R&D systems (Minneapolis, MN, USA). Antibodies against phosphor-VEGFR2 (p-VEGFR2, Y1175), VEGFR2, Akt, p-Akt (S473), Caspase (cysteine-aspartic proteases) 9, and p-BAD (Cell Signaling Technology; Danvers, MA, USA). Aflibercept (40 μg/μL) and ranibizumab (10 μg/μL) were from the pharmacy of Massachusetts Eye and Ear (Boston, MA, USA). The primary antibody against β-actin and secondary antibodies of the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enhanced chemiluminescent substrate for detection of HRP was obtained from Thermo Fisher Scientific (Waltham, MA, USA).
9
Western Blotting for Protein Expression Analysis
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Western blotting was performed as described previously [25 (link)]. Tissue or cells were lysed using RIPA lysis buffer (Beyotime Biotechnology, P0013C) supplemented with PMSF (Thermo Scientific, 36978), protease inhibitors (Roche, 04693132001) and phosphatase inhibitors (Roche, 04906837001). Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Scientific, cat No. 23225). Western blotting was performed with antibodies against the following: KPNA2 (1:1000, Abclonal, A5012), KPNA1 (1:1000, Abclonal, A1742), KPNA3 (1:1000, Abclonal, A8347), KPNA4 (1:1000, Abclonal, A2026), KPNB1 (1:1000, Abclonal, A8610), β-ACTIN (1:1000, CST, 3700S), STAT3 (1:1000, Abclonal, A19566), P-STAT3 (1:1000, Abclonal, AP0705), VEGF (1:1000, Proteintech, 66828–1-Ig), P-VEGFR2 (1:1000, Cell Signaling Technology, #3770), GAPDH (1:1000, Abclonal, AC001), Laminb1 (1:1000, Cell Signaling Technology, #13435), ANGPT2 (1:1000, Abclonal, A11306), Flag (1:1000, Sigma, F2555), JAK1 (1:1000, Abclonal, A18323), JAK2 (1:1000, Abclonal, A7694), SRC (1:1000, Abclonal, A0324), TYK2 (1:1000, Abclonal, A2128), P-JAK1 (1:1000, Abclonal), GPX1 (1:1000, Abclonal, A11166), and MOV10 (1:1000, Abclonal, A7227).
Relative quantitative analysis of protein expression levels was performed using Image Lab software (Bio-Rad).
Relative quantitative analysis of protein expression levels was performed using Image Lab software (Bio-Rad).
10
Western Blotting of Retinal Proteins
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