Four ovarian cancer cell lines, OVCAR3, OVCAR5, IGROV-1 and SKOV3, were used in this study. The cells were grown in DMEM/F12(1:1) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100U/ml penicillin and 100 ug/ml streptomycin under 5% CO2. ONC201 was kindly provided by Oncoceutics, Inc. MTT, propidium iodide and RIPA buffer were purchased from Sigma (St. Louis, MO). Antibodies to PERK (#5683), ATF4 (#11815), CHOP (#2895), BCL-XL (#2764), MCL-1 (#5453), PARP (#9542), DR5 (#8074), β-actin (#3722), VEGF-C (#2445), Slug (#9585), Snail (#3879), CyclinD1 (#2978), CDK4 (#12790), and CDK6 (#3136) were obtained from Cell Signaling Technology (Beverly, MA). DRD2 (B-10, sc-5303) and DRD5 (E-12, sc-376088) were purchased from Santa Cruz Biotechnology. The TMRE (#22220) and JC-1 (#22200) probes were purchased from AAT bioquest (Sunnyvale, CA).
Vegf c
Manufactured by Cell Signaling Technology
Sourced in United States
VEGF-C is a recombinant protein produced by Cell Signaling Technology. It is a member of the vascular endothelial growth factor (VEGF) family and plays a role in the regulation of angiogenesis and lymphangiogenesis.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (3 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
Snail, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
Ecl plus kit, by Thermo Fisher Scientific (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Pro prep, by iNtRON Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
6 protocols using vegf c
1
ONC201 Modulates Ovarian Cancer Signaling
2
CDDP and IGF-1 Signaling in UBC Cells
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UBC cells were treated with CDDP (6 µg/ml), with or without IGF-1 (50 ng/ml), and then lysed with lysis buffer supplemented with protease inhibitor (catalog no. P0013B; Beyotime Institute of Biotechnology, Haimen, China). Western blotting was performed as previously described (10 (link)). In the present study, antibody against HMGN5 (dilution, 1:1,000; catalog no. ab18601) was obtained from Abcam (Cambridge, MA, USA), while antibodies against Akt (catalog no. 4685S), phosphorylated (p)-Akt (Ser473; catalog no. 4060P), slug (catalog no. 9585P), E-cadherin (catalog no. 3195S), VEGF-C (catalog no. 2445S), cytochrome c (catalog no. 19940S), cleaved-caspase-3 (catalog no. 9664P), cleaved-PARP (catalog no. 5625P) and β-actin (catalog no. 4967; all 1:1,000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The proteins were visualized using the ECL Plus Kit (Thermo Fisher Scientific, Inc.). β-actin served as a loading control. Experiments were performed in triplicate.
3
Protein Expression Analysis in Cellular Signaling
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Cells were washed cold PBS and lysed on ice with protein extraction buffer (Pro-Prep, iNtRON Biotechnology, Seoul, Korea). Equal amounts of protein were loaded onto a sodium dodecyl sulfate-polyacrylamide gel (12% polyacrylamide) and transferred to polyvinylidene floride (PVDF) membrane. The membranes were blocked with 5% nonfat milk in TBS-T and incubated with appropriate concentrations of primary antibodies anti-TGFβ receptor II (TβR2), Smad3, P-Erk, P-Akt, Akt, P-Rho, Rho, VEGF-C (dilution 1:1000; cell signaling Technology, Massachusetts, USA) and anti-β-actin (dilution 1:1000; Sigma-Aldrich, USA) antibodies were diluted in TBS-T (TBS/Tween 20: 2% skim milk). The appropriate secondary antibodies were applied (dilution 1:5000, horseradish peroxidase-conjugated anti-rabbit and anti-mouse) at room temperature for 1 h. Labeled bands were detected by enhanced chemiluminescence (ECL; ThermoScientific, USA).
4
Protein Expression Analysis in Larval Homogenates
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Whole larval homogenates from 3 different pools of 15–20 larvae per group, were electrophoresed and transferred to PVDF as previously described [84 ]. Briefly, 7 mg of each protein sample was separated using 4% stacking and 10% separating sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto a filter using a mini trans-blot electrophoretic transfer cell (Bio-Rad, Milan, Italy). The transfer was carried out for 30 min using Trans-Blot TurboTM Transfer System. After blocking in 2% bovine serum albumin (BSA; Merck KGaA, Darmstad, Germany, # A2153 ) in PBS. The following primary antibody was used: LC3A/B (Cell Signaling, Beverly, MA, USA, #BK4108S), Caspase 3 (Cell Signaling, Beverly, MA, USA, # BK9661S), IL6 (Abcam, Cambridge, UK, #ab208113,) and VEGF-C (Cell Signaling, Beverly, MA, USA, # BK2445S,) antibodies were diluted 1:1000 in a solution containing 2% BSA and 0.1% TWEEN 20 in PBS and incubated overnight at 4 °C. β-actin antibody (Cell Signaling, Beverly, MA, USA, # BK4967S) was used as internal standard. The reaction was visualized with ECL-PLUS (GE Healthcare, Milano, Italy) chemiluminescent reagent for Western blotting. Densitometric analysis was performed using Fiji software for Windows.
5
Western Blot Analysis of Cell Signaling
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Cells were harvested and homogenized with lysis buffer (Beyotime Institute of Biotechnology) 48 h post-transfection. Protein concentrations were measured using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Proteins (between 30 and 50 µg) were separated by SDS-PAGE (10% gel) and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Subsequent to blocking with 5% skimmed milk powder at room temperature for 2 h, the membranes were incubated with primary antibodies specific to MTDH (catalog no., 14065S), vascular endothelial growth factor C (VEGF-C; catalog no., 2445S), cyclin D1 (catalog no., 2978T), epithelial (E-) cadherin (catalog no., 3195S) and β-actin (catalog no., 3700S) (all dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA). The membranes were then incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse secondary antibody for β-actin; dilution, 1:5,000; catalog no., sc-2005 or mouse anti-rabbit secondary antibody for MTDH, VEGF-C, cyclin D1 and E-cadherin; catalog no., sc-2357; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The resulting immunoblots were quantified using ImageJ2x software (National Institutes of Health, Bethesda, MD, USA).
6
Antibody Procurement for Protein Analysis
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Antibodies for VEGF-C, P65, p-P65, IκBα, p-IκBα, IKKα, p-IKKα, P38, p-P38, JNK, and p-JNK were purchased from Cell Signaling Technology (Danvers, MA, USA), Antibodies for p-ERK (E4) and ERK 1 (K-23) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), LYVE-1, histone 3, and D2-40 from Abcam (Cambridge, Massachusetts, USA) and β-actin from Sigma-Aldrich (Saint Louis, Missouri, USA), PEDF antibody from Millipore (Bedford, MA, USA), and IL-1β recombinant protein were from Sigma-Aldrich.
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