P flk1

Manufactured by Santa Cruz Biotechnology

P-Flk1 is a laboratory product offered by Santa Cruz Biotechnology. It is a protein that plays a role in the regulation of vascular endothelial growth factor receptor 2 (VEGFR2) signaling. The core function of P-Flk1 is to serve as a tool for researchers to study cellular signaling pathways involved in angiogenesis and vascular development.

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Lab products found in correlation

Cell lysis buffer, by Thermo Fisher Scientific (2 mentions) β actin, by Merck Group (2 mentions) Complete mini protease inhibitor tablet, by Roche (2 mentions) Vegfr2, by Santa Cruz Biotechnology (1 mentions) Horseradish peroxidase conjugated anti rabbit or anti mouse igg, by Jackson ImmunoResearch (1 mentions) Gel doc 2000 imager, by Bio-Rad (1 mentions) Hif 1α, by Santa Cruz Biotechnology (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Horseradish peroxidase conjugated igg, by Jackson ImmunoResearch (1 mentions) P akt, by Cell Signaling Technology (1 mentions) Vegfr2, by Cell Signaling Technology (1 mentions) P akt1 2 3, by Santa Cruz Biotechnology (1 mentions) Phospho p44 42 mapk erk, by Cell Signaling Technology (1 mentions) Phospho pkc, by Cell Signaling Technology (1 mentions) Western lightning plus ecl, by PerkinElmer (1 mentions)

4 protocols using p flk1

Quantification of VEGFR2 and p-Flk1 in ECs

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After 24 hr co-culture, proteins of ECs in different groups were extracted with cell lysis buffer (Thermo Fisher scientific) supplemented with complete mini protease inhibitor tablet (Roche). Then, the protein lysates were electrophoresed through SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 hr at room temperature, and incubated with primary antibody against VEGFR2 (1:1000; Santa Cruz), p-Flk1 (1:1000; Santa Cruz) or β-actin (1:4000; Sigma) at 4°C overnight. On the next day, membranes were washed and incubated with horseradish-peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:40000; Jackson Immuno Research Lab) for 1 hr at room temperature. Blots were developed with enhanced chemiluminescence developing solutions and images were quantified under ImageJ software. The experiment was repeated four times.

Sun X., Ma X., Wang J., Zhao Y., Wang Y., Bihl J.C., Chen Y, & Jiang C. (2017). Glioma stem cells-derived exosomes promote the angiogenic ability of endothelial cells through miR-21/VEGF signal. Oncotarget, 8(22), 36137-36148.

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Protein Expression Analysis in Glioma

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Protein extraction and Western blotting were done as described [48 (link)]. Briefly, proteins from C6 glioma cells cultured in various conditions and glioma tissues were extracted and subjected to SDS/PAGE. Proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies for HIF-1α, Notch1, Flk1, p-Flk1 and GAPDH (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA), respectively. After incubation with secondary antibodies, the proteins were detected by enhanced chemiluminescence and quantified using a Gel Doc 2000 Imager (Bio-Rad, Hercules, CA). Each experiment was replicated three times.

Chen X., Fang J., Wang S., Liu H., Du X., Chen J., Li X., Yang Y., Zhang B, & Zhang W. (2014). A new mosaic pattern in glioma vascularization: exogenous endothelial progenitor cells integrating into the vessels containing tumor-derived endothelial cells. Oncotarget, 5(7), 1955-1968.

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Evaluating EPC-NPC Crosstalk in Hypoxia-Injured Endothelial Cells

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H/R-injured ECs were harvested after co-cultured with EPCs and/or NPCs. Proteins were extracted with cell lysis buffer (Thermo scientific) supplemented with complete mini protease inhibitor tablet (Roche). Protein lysates were electrophoresed through SDS-PAGE gels and transferred onto PVDF membranes. The membranes were blocked with 5 % non-fat milk for 1 h and incubated with primary antibodies against Akt (1:1000; Cell Signaling), p-AKt (1:1000; Cell Signaling), VEGFR2 (1:1000; Cell Signaling), p-Flk1 (1:200; Santa Cruz), and

β

-actin (1:4000; Sigma) at 4 °C for overnight. After washing, membranes were incubated with horseradish-peroxidase-conjugated IgG (Jackson Immuno Research Lab) for 1 h at RT. Blots were developed with enhanced chemiluminescence developing solutions and quantified under ImageJ software. For detecting the protein expressions of Akt and p-AKt in all groups, two sets of gels were done. One set of gels was used to compare the difference between normoxia and hypoxia groups, and the other set of gels was used to compare the differences among different treatment groups in the Hypoxia groups. All experiments were repeated at least six times. Similarly, Es was calculated as: Es = (E_{EPC + NPC} − E_EPC − E_NPC) / (E_EPC +E_NPC) x 100 %.

Wang J., Chen Y., Yang Y., Xiao X., Chen S., Zhang C., Jacobs B., Zhao B., Bihl J, & Chen Y. (2016). Endothelial progenitor cells and neural progenitor cells synergistically protect cerebral endothelial cells from Hypoxia/reoxygenation-induced injury via activating the PI3K/Akt pathway. Molecular Brain, 9, 12.

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Lung Explant Hypoxia Signaling Assay

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At least twelve lung explants/condition at E11.5 or post caval lobes at E15.5 were cultured in 3% and 50% oxygen for 2 days followed by homogenization in RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 20 mM Tris, 0.16 M NaCl, 1 mM EGTA, 1 mM EDTA, 15 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 0.5 μg/mL leupeptin), and 0.5 μg/mL pepstatin A containing Proteinase Inhibitor Complete and phosphatase inhibitor (Roche Molecular Biochemicals, IN). Lysates were centrifuged at 10,000 rpm for 30 minutes at 4°C and supernatants used as whole tissue lysates. Protein concentration was measured using the Bradford assay. Equal amounts of protein (50–70 micrograms) were separated on NuPAGE 4-12% Bis–Tris Gels and transferred to nitrocellulose, as described earlier. Western blots were performed with antibodies against p-FLK1 (Tyr 996)(1:200 Santa Cruz Biotechnology, CA), phospho-PKC (1:1000 Cell Signaling Technologies), p-Akt 1/2/3 (1:500, Santa Cruz Biotechnology, CA) and phospho-p44/42 MAPK (Erk ½) (1:1000 Cell Signaling Technologies). To ensure equal loading, membranes were re-probed with and antibody to PKC, Akt and Actin (I-19) (1:2000 Santa Cruz Biotechnology, CA). Specific bands were visualized after incubation with the respective secondary antibodies by autoradiography by using Enhanced Chemiluminescence Substrate (Western Lightning Plus-ECL, PerkinElmer Inc, MA).

Esquibies A.E., Karihaloo A., Quaggin S.E., Bazzy-Asaad A, & Cantley L.G. (2014). Heparin binding VEGF isoforms attenuate hyperoxic embryonic lung growth retardation via a FLK1-neuropilin-1-PKC dependent pathway. Respiratory Research, 15(1), 32.

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