Cells were lysed using a RIPA lysis buffer (Sigma). Equal amounts of protein from each sample were loaded onto the SDS-PAGE and then transferred to PVDF membranes (Thermo Fisher Scientific, Waltham, MA USA). After blocking with 5% nonfat milk in PBST for 1 h at room temperature, membranes were exposed to ARF6 (Santa Cruz Biotechnology, Inc Dallas, Texas), NEDD9 (Abcam, Cambridge, MA), MT1-MMP (Abcam.), RAB-8 (Cell Signaling Technology, Danvers, MA), Rho A, cortactin, WIP, paxcillin (Santa Cruz Biotechnology) NWASP (EMD MiIllipore, Billerica, MA) and β-actin (Sigma-Aldrich) antibodies. The enhanced chemiluminescence system was used to visualize the protein bands as recommended by the manufacturer (Thermo Fisher Scientific). Protein bands were quantified using densitometry software (Bio-Rad), and normalized using actin as a loading control. To calculate the relative intensity of each band, individual bands were divided by the corresponding loading control intensity.
Nedd9
Manufactured by Abcam
Sourced in United States
NEDD9 is a protein that plays a role in cell adhesion and migration. It functions as an adaptor protein, facilitating the interaction between various signaling molecules involved in these cellular processes. NEDD9 is expressed in a variety of cell types and is implicated in various biological pathways.
Automatically generated - may contain errors
Lab products found in correlation
N cadherin, by Santa Cruz Biotechnology (2 mentions)
Pan keratin, by Cell Signaling Technology (2 mentions)
P130cas, by Santa Cruz Biotechnology (2 mentions)
Psrcy527, by Cell Signaling Technology (2 mentions)
Sv40 t ag, by Santa Cruz Biotechnology (2 mentions)
P stat3 y705, by Cell Signaling Technology (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
E cadherin, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Cortactin, by Santa Cruz Biotechnology (1 mentions)
Densitometry software, by Bio-Rad (1 mentions)
Mt1 mmp, by Abcam (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Sox10, by Abcam (1 mentions)
Delta vision imaging system, by Cytiva (1 mentions)
6 protocols using nedd9
1
Western Blot Analysis of Cytoskeletal Proteins
2
Immunofluorescence Analysis of Colon Cancer
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Colon cancer cells treated by Ptero (40 µM) and/or MLT (1 mM) for 24 h were fixed with 4% formaldehyde and were permeabilized in 0.1% Triton X-100, according to the paper [13 (link)]. The fixed cells were washed with 1X PBS, blocked with 2% BSA in 1X PBS for 30 min at room temperature (RT), and incubated with the specific antibodies of NEDD9, SOX10, and Ki67 (1:1000; Abcam, Cambridge, UK) overnight at 4 °C. After washing, the cells were incubated with Alex Fluor 489 goat mouse-IgG antibody (Invitrogen) and Alexa Fluor 546 goat rabbit-IgG antibody (1:1000) for 1 h at RT. After washing twice, the nuclei of the cells were stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma) and then were visualized under a FLUOVIEW FV10i confocal microscope (Olympus). Images of NEDD9- and SOX10-stained cells were taken by a Delta Vision imaging system (Applied Precision, Issaquah, WA, USA).
3
Western Blot Analysis of DNA Damage Signaling
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The cells were lysed in PierceTM RIPA Buffer (ThermoScientific, Waltham, MA, USA). The protein concentrations were established using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Western blotting was performed using standard procedures, and the blots were developed using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). Original images of western blot added to the Supplementary Data . Primary antibodies used were as follows: ERCC4 (#13465), p-ATM (Ser1981) (#13050), p-ATR (Ser428) (#2853), p-Chk1 (Ser345) (#2341), p-Chk2 (Thr68) (#2197), p-CDC25C (Ser216) (#9528), p-H2AX (Ser139) (#3257000) (1:1000; Cell Signaling Technology, Danvers, MA, USA); NEDD9 (ab18056, 1:1000), beta-Actin (ab49900, 1:100,000) (Abcam; Cambridge, MA, USA), Vinculin (700062) (1:1000, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Vinculin and beta-Actin were used as the loading controls. The quantification was performed using NIH ImageJ Imaging Software (Rayne Rasband, National Institutes of Health, USA).
4
Western Blot Analysis of CAPAN1 Cells
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Forty-eight hours after transfection, the CAPAN1 cells were lysed in RIPA buffer (Thermo Fisher Science, Waltham, MA, USA) and the cellular protein concentrations were determined with BCA kit (Beyotime, Nanjing, China). The proteins resolved on an SDS denatured polyacrylamide gel were transferred onto a nitrocellulose membrane (Millipore, Burlington, MA, USA). The membranes were incubated with diluted primary antibodies (anti-Bax, caspase 3, PI3K, AKT, NEDD9 and β-actin, Abcam, USA) overnight at 4°C. The membranes were washed and incubated with goat anti-mouse secondary antibodies (1:10,000) and visualized using an ECL kit (Millipore).
5
Derivation and Characterization of Ovarian Cancer Cell Lines
6
Derivation and Characterization of Ovarian Cancer Cell Lines
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MOVCAR cell lines were derived from the ascites of MISIIR-TAg;NEDD9+/+ (MOVCAR-5009, -6111, -7577, -8248, -8250) and MISIIR-TAg;NEDD9−/− (MOVCAR-136, -143, -145, -168) mice in our laboratory as described12 . Human OC cell lines OVCAR-5 and A1847 OC were obtained from the FCCC Cell Culture Facility (deposited by Dr. Thomas Hamilton) and maintained as described56 (link). Cell lines were periodically checked for mycoplasma contamination OVCAR-5 cells were authenticated by STR profiling. Antibodies and commercial source: antibodies recognizing AKT (cat# 468S), pAKTS473 (cat# 3787S), ERK 1/2 (9102), pERK1/2T202-Y204 (cat# 9106S), Src (cat# 2108S), pSrcY416 (cat# 2101S), pSrcY527 (2105S), STAT3 (cat# 9132) and pSTAT3Y705 (cat# 9145S), pan-keratin (cat# 4545) (Cell Signaling Technology; Danvers, MA, USA), NEDD9 (cat# ab18056) (Abcam; Cambridge, MA, USA), SV40-TAg (cat# sc-147), β-actin (cat# sc-8432), E-cadherin (cat# sc-7870), N-cadherin (cat# sc-7939), p130Cas (cat# sc-860) (Santa Cruz Biotechnology; Santa Cruz, CA, USA), BMPR1B (cat# bs-6639R), and FOXJ1 (cat# 36887) (One World Lab; San Diego, CA, USA), and PAX8 (cat# 10336-I-AP) (Proteintech Group, Rosemont, IL, USA).
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