After one wash with PBS, cells were incubated with appropriate dilutions of various combinations of the following antibodies for 30 min. Primary antibodies to cell surface markers directed against CD14 (17‐0149‐42), CD16 (12‐0167‐42), and BEST1 (PA5‐77290) were purchased from Invitrogen, VEGFR1 (13 687), and VEGFR2 (26 415) were purchased from Proteintech. The stained cells were acquired by a BD LSR II Flow Cytometer (BD Biosciences), and data generated were processed using FlowJo software.
Vegfr2
Manufactured by Proteintech
Sourced in China
VEGFR2 is a receptor tyrosine kinase that plays a crucial role in angiogenesis and vascular development. It is a member of the vascular endothelial growth factor (VEGF) receptor family and is expressed primarily on endothelial cells. VEGFR2 is responsible for transducing signals from VEGF, which is essential for the proliferation, migration, and differentiation of endothelial cells during the formation of new blood vessels.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Sp0041, by Solarbio (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Phospho iκbα ser32, by Cell Signaling Technology (1 mentions)
Phospho p65 ser536, by Abcam (1 mentions)
Ecl western blotting kit, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cd133, by Proteintech (1 mentions)
Alexa fluor 594 conjugated secondary antibody, by Proteintech (1 mentions)
Coralite488 conjugated secondary antibody, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Goat serum, by Solarbio (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Fibronectin, by Proteintech (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
P egfr, by Proteintech (1 mentions)
P jak1, by Proteintech (1 mentions)
6 protocols using vegfr2
1
Multicolor Flow Cytometry Immunophenotyping
2
Immunohistochemical Analysis of VEGF and CD31
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Cells were seeded on 24-well slides and processed according to the experimental protocol in cell immunohistochemistry experiments. At the indicated time points, cells were washed three times with PBS, then fixed with 4% paraformaldehyde. After blocking the cells with goat serum for 30 min, the cells were incubated with the corresponding primary antibodies (VEGFa, #19003-1-AP and CD31, #28083-1-AP, Proteintech, China) at 4 °C overnight. Then, cells were incubated with secondary antibody for 1 h at room temperature and stained with 4′,6-diamidino-2-phenylindole (DAPI).
For section immunohistochemistry experiments, sections were deparaffinized using xylene, rehydrated with graded ethanol, then treated with 3% hydrogen peroxide for 10 min, and boiled in antigen retrieval solution at 95–100 °C for 20 min, and blocked with normal goat serum for 30 min. Tissue sections were incubated with corresponding primary antibodies (VEGFR2, #26415-1-AP and CD31, #28083-1-AP, proteintech, China) overnight at 4 °C. After three washed with PBS, sections were incubated with biotin-labeled secondary antibody (SP0041, Solarbio, China) for 30 min, followed by streptavidin–horseradish peroxidase (HRP) conjugate for 20 min at room temperature. Staining without primary antibody was used as a negative control. Immunohistochemistry results were quantified using Image J software.
For section immunohistochemistry experiments, sections were deparaffinized using xylene, rehydrated with graded ethanol, then treated with 3% hydrogen peroxide for 10 min, and boiled in antigen retrieval solution at 95–100 °C for 20 min, and blocked with normal goat serum for 30 min. Tissue sections were incubated with corresponding primary antibodies (VEGFR2, #26415-1-AP and CD31, #28083-1-AP, proteintech, China) overnight at 4 °C. After three washed with PBS, sections were incubated with biotin-labeled secondary antibody (SP0041, Solarbio, China) for 30 min, followed by streptavidin–horseradish peroxidase (HRP) conjugate for 20 min at room temperature. Staining without primary antibody was used as a negative control. Immunohistochemistry results were quantified using Image J software.
3
Western Blot Analysis of NF-κB Signaling
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Lung tissues were minced and lysed in lysis buffer. The protein concentrations in the supernatants were detected using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Next, equivalent amounts of proteins from each sample were separated using sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore). After blocking with 5% bovine serum albumin (Sigma) for 1 h, the membranes were incubated with their respective primary antibodies, including antibodies against phospho-P65 (Ser536) (1:300; Abcam, UK), P65 (1:500; Proteintech Group), phospho-IκBα (Ser32) (1:500; Cell Signaling Technology), IκBα (1:500; Proteintech Group), and VEGFR2 (1:500; Proteintech Group), overnight at 4°C. The membranes were then incubated with a secondary antibody at room temperature for 1 h. Blots were visualized using the ECL Western Blotting Kit (Millipore), and the results were subjected to gray value analysis using Quantity One analysis software.
4
Immunofluorescent Staining of Endothelial Markers
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The cells were washed with PBS, fixed with 3–4 ml of 4% paraformaldehyde for 30 min at 4 °C, washed with PBS three times and finally permeabilized with a mixture of 10% goat serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and 0.5% Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The cells were immunofluorescently stained with CD31 (Proteintech Group, Inc., Chicago, USA), CD34 (Beijing Bioss Molecular Co., Ltd., Beijing, China), CD133 (Proteintech Group, Inc., Chicago, USA), CD144 (Beijing Bioss Molecular Co., Ltd., Beijing, China) and VEGFR2 (Proteintech Group, Inc., Chicago, USA) antibodies, which were diluted to 100 ml, overnight at 4 °C. The cells were then washed with PBS three times (5 min/time) and incubated with CoraLite 488-conjugated secondary antibody (Proteintech Group, Inc., Chicago, USA) and Alexa Fluor 594-conjugated secondary antibody (Proteintech Group, Inc., Chicago, USA) in the dark at room temperature for 2 h. The cell DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The cells were examined by fluorescence microscopy (IX71, Olympus Corporation, Shibuya, Tokyo, Japan).
5
Proteomic Analysis of Signaling Pathways
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Cells were lysed on ice using lysis buffer as instructed before27 ; then the lysate proteins were collected and further analyzed. Protein A/G agarose or Ni‐NTA beads were used for immunoprecipitation or pulldown assays, respectively. Immunoblotting was performed according to standard method with primary antibodies against pY antibody (Abcam EPR16871 ), pEGFR (CST#3777S), EGFR (CST#4267), pSTAT3 (CST#4113), STAT3 (CST#9139), CK8 (17514‐1‐AP, Proteintech), CD71 (ABclonal#A5865), Vimentin (10366‐1‐AP, Proteintech), GRB2 (CST#3972), Snail1 (ABclonal#A5243), pIGF‐1R (CST#3021), IGF‐1R (20254‐1‐AP, Proteintech), IRS‐1 (17509‐1‐AP, Proteintech), pVEGFR2 (CST#3817S), VEGFR2 (CST#9698), SHC (10054‐1‐AP, Proteintech), pAKT (CST#4060), pERK1/2 (CST#4370), pJAK1 (CST#3331), AKT (CST#9272), N‐cadherin (22018‐1‐AP, Proteintech), ERK1/2 (CST#4695), JAK1 (CST#3344), LIFR (22779‐1‐AP, Proteintech), His (CST#12698), GP130 (21175‐1‐AP, Proteintech), αSMA (55135‐1‐AP, Proteintech), E‐cadherin (20874‐1‐AP, Proteintech), Smad2/3 (CST#8685), Fibronectin (15613‐1‐AP, Proteintech), FAP (ABclonal#A6349), collagen‐I (14695‐1‐AP, Proteintech), pSmad2/3 (CST#8828), TWIST1 (25465‐1‐AP, Proteintech), and GAPDH (CST#5174) were used at recommended dilutions followed by HRP‐conjugated antibodies and analyzed with the ECL system.
6
Protein Expression Analysis of Stem Cells
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Cells were collected and followed by lysing in RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors. Protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, WI, USA). Total protein (40-60 µg per sample) was then loaded for sodium dodecyl sulfate gel electrophoresis and transferred onto polyvinylidene uoride membranes (Millipore, Billerica, MA, USA).
The membranes were incubated with relevant primary antibodies overnight at 4 °C, followed by the horseradish peroxidase-conjugated secondary antibodies (ZSGB-BIO, Beijing, China). Glyceraldehyde 3phosphate dehydrogenase (GAPDH) was served as the loading control. Antibodies for CD133, CD44, Oct4, Sox2, Nanog, epithelial cellular adhesion molecule (EpCAM), P glycoprotein(P-gp), ABCC1, VEGFR-2, SHH, Smo, Gli1, Gli2, PCNA, Cyclin D1, Bcl-2, Bax, Cleaved Caspase (8,9,3) and GAPDH were purchased from Proteintech (Rosemont, IL, USA).
The membranes were incubated with relevant primary antibodies overnight at 4 °C, followed by the horseradish peroxidase-conjugated secondary antibodies (ZSGB-BIO, Beijing, China). Glyceraldehyde 3phosphate dehydrogenase (GAPDH) was served as the loading control. Antibodies for CD133, CD44, Oct4, Sox2, Nanog, epithelial cellular adhesion molecule (EpCAM), P glycoprotein(P-gp), ABCC1, VEGFR-2, SHH, Smo, Gli1, Gli2, PCNA, Cyclin D1, Bcl-2, Bax, Cleaved Caspase (8,9,3) and GAPDH were purchased from Proteintech (Rosemont, IL, USA).
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