Chemical reagents were obtained from Sigma Chemical Company (St. Louis, MO, USA) and HiMedia Labs (Mumbai, India). Nimbolide was purchased from Asthagiri Herbal Research Foundation, Chennai. Antibodies for GAPDH, TIMP-2, RECK, MMP-2, MMP-9, VEGF, VEGFR2 and HIF1α were purchased from Santa Cruz Biotechnology, USA. p-VEGFR2Tyr1175, Notch-1, NICD, ADAM-10 and histone (H2B) antibodies were from Cell Signaling Technology, USA. Fetal bovine serum was from GIBCO, Invitrogen, NY, USA. Power SYBR Green PCR master mix was obtained from Applied Biosystems, California, USA. FuGENE transfection reagent was procured from Promega. Oligonucleotide primers were procured from Sigma Genosys, San Ramon, USA. All other reagents used were of analytical grade.
P vegfr2 tyr1175
Manufactured by Cell Signaling Technology
Sourced in United States
P-VEGFR2(Tyr1175) is a phospho-specific antibody that recognizes the phosphorylated form of VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) at tyrosine 1175. This antibody is used to detect and quantify the activation of VEGFR2, which is a key receptor involved in angiogenesis and vascular development.
Automatically generated - may contain errors
Lab products found in correlation
Vegfr2, by Cell Signaling Technology (2 mentions)
Fugene transfection reagent, by Promega (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (1 mentions)
Notch1, by Cell Signaling Technology (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Chemical reagents, by Merck Group (1 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Mmp 2, by Santa Cruz Biotechnology (1 mentions)
Vegfr2, by Santa Cruz Biotechnology (1 mentions)
Adam10, by Cell Signaling Technology (1 mentions)
Histone h2b, by Cell Signaling Technology (1 mentions)
Hif 1α, by Santa Cruz Biotechnology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Recombinant human vegf, by Thermo Fisher Scientific (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Total vegfr2, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Total erk1 2, by Cell Signaling Technology (1 mentions)
6 protocols using p vegfr2 tyr1175
1
Nimbolide Inhibits Angiogenic Signaling
2
Structural Modification and Evaluation of Asiatic Acid Derivatives
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AA was extracted from C. asiatica (L.) Urban and structurally modified in our laboratory to produce AA-PMe.20 (link) AA and AA-PMe were both dissolved in 1% dimethyl sulfoxide (DMSO; Sigma-Aldrich Co, St Louis, MO, USA) and their structures are presented in Figure 1 . Endogenous alkaline phosphatase (EAP) staining was tested by a phosphatase substrate kit (Pierce; Thermo Fisher Scientific, Waltham, MA, USA). Recombinant human VEGF was purchased from Thermo Fisher Scientific. Primary antibodies for total Akt, pAkt-ser473, total ERK1/2, p-pERK1/2, total VEGFR2, and pVEGFR2-Tyr1175 were brought from Cell Signaling Technology (Danvers, MA, USA). GAPDH and tubulin antibody were from Abcam (Cambridge, UK).
3
Quantitative Protein Analysis in Cells
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Protein content from all samples was quantified using a bicinchoninic acid protein assay kit (23225; Thermo Fisher). Western blot analysis was performed using standard procedures, was detected using an enhanced chemiluminescence western blotting detection kit (32106; Thermo Fisher), and was quantified by scanning densitometry. Antibodies against phosphorylated (p)-protein kinase B (Akt) (Ser473) (#4060), Akt (#4685), p-extracellular signal-regulated kinase (ERK) (Thr202/Tyr204) (#4370), ERK (#4695), VEGF receptor-2 (VEGFR2, #9698), p-VEGFR2 (Tyr1175) (#3770), β-actin (#4970), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). CD36 (18836), CPT1A (15184), carnitine palmitoyl-transferase 1B (CPT1B; 22170), VEGF (19003), apolipoprotein A1 (APOA1; 14427), and golgi matrix protein 130 (GM130; 11308) were purchased from Proteintech. AGPAT1 (GTX55496) was purchased from GeneTex. CD31 (ab213175), tumor susceptibility 101 (TSG101, ab125011), and CD81 (ab109201) were purchased from Abcam. GAPDH or β-actin was used as a loading control.
4
Protein Expression Analysis in CRC Cells
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Total proteins were extracted from human CRC cells or human and mouse colonic tissues as described elsewhere [42 (link)] and separated by SDS/PAGE. Blots were incubated with antibodies to IL-21 (Millipore, Milan, Italy), p-STAT3 Tyr705, p-NF-κB/p65 Ser536, p-VEGF-R2 Tyr1175, VEGF-R2 (all from Cell Signaling, Danvers, MA), STAT3, p65 and IL-21R antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA). To ascertain equivalent loading of the lanes, blots were stripped and incubated with an anti-β-actin antibody.
5
Apatinib Modulates Protein Expression
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Western blot analyses were carried out to determine the expression levels of various proteins. Cells were pre-treated with apatinib at different concentrations or DMSO (diluent) for 48 h. Next, the cells were harvested, washed with cold 1 × PBS, and lysed in moderate RIPA lysis buffer (Beyotime Biotechnology) containing a protease inhibitor cocktail (Roche Diagnostics). Total protein concentrations were determined using a BCA protein assay kit (Beyotime Biotechnology). Equal amounts (24 μg per load) of the protein samples were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The resulting blots were blocked in 5% bovine serum albumin (BSA) and incubated with primary antibodies, followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP). Protein bands were visualized using a chemiluminescent reagent (Millipore). Antibodies directed against LDHA, HK2, SOX5, VEGFA, AKT1 and β-Actin were purchased from Proteintech, and antibodies directed against VEGFR2, p-AKT1(Ser473), p-GSK3β (Glycogen synthase kinase 3 beta) (Ser9) and p-VEGFR2(Tyr1175) were purchased from Cell Signaling Technology. The antibody directed against GLUT4 was obtained from Signalway Antibody.
6
Immunohistochemistry analysis of xenograft tumors
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Immunohistochemistry (IHC) assays were carried out as described previously [23, 24] . IHC was performed on xenograft tumor sections using antibodies directed against p-VEGFR2(Tyr1175) (rabbit polyclonal antibody, Cell Signaling Technology, 1:100 dilution), p-AKT1(Ser473) (rabbit polyclonal antibody, Cell Signaling Technology, 1:100 dilution), p-GSK3β(Ser9) (rabbit polyclonal antibody, Cell Signaling Technology, 1:300 dilution), CD31 (rabbit polyclonal antibody, Abcam, 1:50 dilution), GLUT4 (rabbit polyclonal antibody, Signalway Antibody, 1:100 dilution) and SOX5 (rabbit polyclonal antibody, Proteintech, 1:100 dilution). IHC staining was scored based on both the percentage of positive tumor cells and staining intensity. Negative expression was defined as < 2+ and positive as > 2+ to < 6 + .
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