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Anti phospho fak y397

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Anti-phospho-FAK (Y397) is a primary antibody that detects the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397. This antibody can be used to study the activation and signaling of FAK, which is important for various cellular processes such as cell adhesion, migration, and proliferation.

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Lab products found in correlation

Anti fak, by Cell Signaling Technology (4 mentions) Anti gapdh, by Cell Signaling Technology (3 mentions) Anti phospho akt s473, by Cell Signaling Technology (2 mentions) Anti vinculin, by Merck Group (2 mentions) Alexa 647, by Thermo Fisher Scientific (2 mentions) Anti rep 1, by Abcam (2 mentions) Anti rabgdi, by Santa Cruz Biotechnology (2 mentions) Anti rabif mouse monoclonal antibody d 12, by Santa Cruz Biotechnology (2 mentions) Anti rab13 rabbit polyclonal, by Novus Biologicals (2 mentions) Rabbit monoclonal anti gapdh 14c10, by Cell Signaling Technology (2 mentions) Rabbit polyclonal anti gfp, by Thermo Fisher Scientific (2 mentions) Anti phospho erk, by Santa Cruz Biotechnology (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Anti β actin, by Cell Signaling Technology (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Endothelial cell growth supplement (ecgs), by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Fitc phalloidin, by Merck Group (1 mentions) Anti s6k, by Abcam (1 mentions) Anti akt1, by Proteintech (1 mentions)

Spelling variants of this product

Anti-FAK (97 citations) Phospho-FAK (30 citations) Anti-phospho-FAK (19 citations) Phospho-FAK (Y397) (7 citations) Y397-FAK (3 citations) Rabbit anti-phospho-FAK (Y397) (3 citations)

12 protocols using anti phospho fak y397

1

mTOR Modulation Impacts Endothelial Angiogenesis

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Medium 199 (M199), Hank's Balanced Salt Solution (HBSS), fetal bovine serum (FBS), and endothelial cell growth supplement were purchased from Invitrogen (Burlington, ON). VEGF-A was from Peprotech (Princeton, NJ). Rapamycin, PP242, a highly specific mTOR active-site inhibitor [12 (link)], and anti-tubulin-α was from Millipore (Temecula, CA). Ku-0063794, a second specific mTOR inhibitor [13 (link)] was from Sigma (St. Louis, MO). Anti-Akt1 was from Protein Tech (Chicago, IL). Hiperfect, non-silencing short interfering RNA (siRNA) and Akt1 silencing siRNA were from Qiagen Inc (Mississauga, ON). Human tumor necrosis factor-α (TNFα) was from Cedarlane (Mississauga, ON). Cycloheximide, phalloidin-FITC, anti-vinculin, and DAPI were from Sigma. Anti-S6K was from Abcam (Cambridge, UK). Anti-phospho-AktS473, anti-phospho-S6KT389, anti-FAK, anti-phospho-FAKY397, anti-raptor, anti-rictor, and rictor siRNA were from Cell Signaling Technology (Danvers, MA). ON-TARGETplus human raptor siRNA-SMARTpool was from Thermo Scientific (Waltham, MA).
Farhan M.A., Carmine-Simmen K., Lewis J.D., Moore R.B, & Murray A.G. (2015). Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis. PLoS ONE, 10(8), e0135245.
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2

HUVEC Cell Squalamine Treatment Regulates FAK Signaling

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HUVEC cells (Lifeline Cell Technologies, Danvers, MA) were grown in 100 mm culture dishes to 80-90% confluence in complete HUVEC cell medium Vasculife (Lifeline Cell Technologies) without VEGF. Before adding VEGF (50 ng/ml) for 10 minutes at 37°C, cells were incubated in the presence or absence of 1 μM squalamine for 1 hour. Appropriate controls were also prepared. After treatment, cells were rinsed with PBS 3 times and placed on ice before lysing in 100 μl cold RIPA buffer (Thermo Fisher Scientific) in the presence of protease inhibitors (Thermo Fisher Scientific). Total protein concentration was determined by a standard Bradford Assay. Protein samples (50 μg/lane) were loaded on a 4-12% precast Tris-Glycine gel . Proteins were then transferred to a PVDF membrane and immunoblotted with anti-phosphoFAK (Y397) and anti-total FAK monoclonal antibodies (Cell Signaling Technology). Bands were detected using the Pierce ECL Western Blotting Substrate blocked with milk (Thermo Fisher Scientific).
Márquez-Garbán D.C., Gorrín-Rivas M., Chen H.W., Sterling C J.r., Elashoff D., Hamilton N, & Pietras R.J. (2019). Squalamine blocks tumor-associated angiogenesis and growth of human breast cancer cells with or without HER-2/neu overexpression. Cancer letters, 449, 66-75.
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3

Western Blot Analysis of Signaling Proteins

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Cell lysates were prepared in ice-cold RIPA lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP40, 0.5% Na-deoxycholate, 0.1% SDS) supplemented with HALTTM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) and sonicated using a Bioruptor® Pico sonication device (diagenode). Protein content was determined with the BCA Protein Assay Kit (Pierce). 20 µg of protein were resolved by SDS–PAGE, transferred to Nitrocellulose membrane (Thermo Fisher Scientific) and blocked in 5% Milk/TBST for 1 h at RT. Primary antibodies were used as follows: anti-p63 (ab735; 1:500; abcam), anti-phospho FAK-Y397 (#8556; 1:500; Cell Signaling Technology), anti-FAK (#3285; 1:1000; Cell Signaling Technology), anti-phospho Src-Y416 (#6943; 1:1000; Cell Signaling Technology), anti-Src (#2109; 1:1000; Cell Signaling Technology), TCF7 (#2203; 1:1000; Cell Signaling Technology), anti-GAPDH (#5174; 1:5000; Cell Signaling Technology), and anti-ACTB (#A2228; 1:1000, Sigma-Aldrich).
Ding L., Su Y., Fassl A., Hinohara K., Qiu X., Harper N.W., Huh S.J., Bloushtain-Qimron N., Jovanović B., Ekram M., Zi X., Hines W.C., Alečković M., Gil del Alcazar C., Caulfield R.J., Bonal D.M., Nguyen Q.D., Merino V.F., Choudhury S., Ethington G., Panos L., Grant M., Herlihy W., Au A., Rosson G.D., Argani P., Richardson A.L., Dillon D., Allred D.C., Babski K., Kim E.M., McDonnell CH I.I.I., Wagner J., Rowberry R., Bobolis K., Kleer C.G., Hwang E.S., Blum J.L., Cristea S., Sicinski P., Fan R., Long H.W., Sukumar S., Park S.Y., Garber J.E., Bissell M., Yao J, & Polyak K. (2019). Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in ductal carcinoma in situ. Nature Communications, 10, 4182.
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4

Immunofluorescence and Western Blot Protocols for Rab13 and Related Proteins

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For IF, cells were plated onto collagen IV‐coated glass coverslips (10 μg/ml) and then fixed with 4% paraformaldehyde (PFA) in PBS (phosphate‐buffered saline) for 15 min, permeabilized with 0.2% Triton X‐100 in PBS for 5 min, blocked with 5% goat serum in PBS for 1 h, and incubated with Rab13 rabbit polyclonal antibody (Novus Biologicals, cat# NBP1‐85799, 1/200 dilution) for 1 h. Secondary antibodies were conjugated with Alexa 647 (Thermo Fisher Scientific).
For Western blot detection, the following antibodies were used: anti‐Rab13 rabbit polyclonal (Novus Biologicals, cat# NBP1‐85799, 1/1,000 dilution), anti‐GFP rabbit polyclonal (Invitrogen, cat# A11122, 1/2,000 dilution), anti‐RhoGDI, anti‐transferrin receptor (clone H68.4, Thermo Fisher Scientific, cat# 13‐6800), anti‐α‐tubulin mouse monoclonal (Sigma‐Aldrich, cat# T6199, 1/10,000 dilution), anti‐GAPDH rabbit monoclonal 14C10 (Cell Signaling Technology, cat# 2118, 1/2,000 dilution), anti‐phospho‐FAK(Y397) (Cell Signaling Technology, cat# 3283), anti‐REP‐1 (Abcam, cat# 134964), anti‐RabGDI (Santa Cruz Biotechnology, cat# sc‐374649), and anti‐RABIF mouse monoclonal antibody D‐12 (Santa Cruz biotechnology, cat# sc‐390759, 1/1,000 dilution).
Moissoglu K., Stueland M., Gasparski A.N., Wang T., Jenkins L.M., Hastings M.L, & Mili S. (2020). RNA localization and co‐translational interactions control RAB13 GTPase function and cell migration. The EMBO Journal, 39(21), e104958.
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5

Western Blot Analysis of Protein Signaling

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Protein was extracted from whole cell lysates using the Mammalian Protein Extraction Reagent M-PER (Thermo Scientific Pierce, Rockford, IL) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO) and quantified through the BCA protein assay (Thermo Scientific Pierce). Lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. We probed for anti-PTEN (Cascade Bioscience clone 6H2.1) at 1:2000, anti-phospho-AKT S473 (Cell Signaling #4060) at 1:1000, anti-phospho-AKT T308 (Cell Signaling #2965) at 1:1000, anti-total AKT (Cell Signaling #9272) at 1:1000, anti-phospho-ERK1/2 (Cell Signaling #9101) at 1:1000, anti-ERK1/2 (Cell Signaling #9102) at 1:1000, anti-phospho-FAK Y397 (Cell Signaling #8556) at 1:1000, anti-phospho-FAK Y576/577 (Cell Signaling #3281) at 1:1000, anti-phospho-FAK Y925 (Cell Signaling #3284) at 1:1000, anti-FAK (Cell Signaling #13009) at 1:1000, and anti-GAPDH (Cell Signaling #2118) at 1:40,000 dilutions. For each experiment, all genotypes were run in parallel on the same gel. Blots were scanned digitally and quantified using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE) and Image J software (NIH, Bethesda, MD). Original blot images are shown in Supplementary Fig. 9.
Yehia L., Ni Y, & Eng C. (2017). Germline TTN variants are enriched in PTEN-wildtype Bannayan–Riley–Ruvalcaba syndrome. NPJ Genomic Medicine, 2, 37.
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6

Implant Surface Characterization Protocols

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Four different implant surfaces (n = 5, each) were used for the study: machined (M), dual acid-etched (DAA), resorbable media microblasted and acid-etched (MBAA), and acid-etch microblasted (AAMB) (Ossean, Intra-Lock International, Boca Raton, FL, USA). All materials were sterilized by exposure to Gamma irradiation. Antibodies: Anti-phospho-Src (Y416), anti-Src, anti-phospho-FAK (Y397), anti-phospho-FAK (Y925), anti-FAK, anti-CDK 6, anti-beta-actin were used (Cell Signaling Technology, Inc., Danvers, MA, USA).
Zambuzzi W.F., Bonfante E.A., Jimbo R., Hayashi M., Andersson M., Alves G., Takamori E.R., Beltrão P.J., Coelho P.G, & Granjeiro J.M. (2014). Nanometer Scale Titanium Surface Texturing Are Detected by Signaling Pathways Involving Transient FAK and Src Activations. PLoS ONE, 9(7), e95662.
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7

Comprehensive Panel of Breast Cancer Cell Lines

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MDA-MB-231, MCF7, T47D, SKBR3, BT474, BT-20, MDA-MB-468, Hs578t and MDA-MB-453 and MDA-MB-157 cells purchased from the American Type Culture Collection (Manassas, VA). MFM 223, SUM159PE and SUM185PE cells were kindly provided by Dr. Jennifer A. Pietenpol (Vanderbilt-Ingram Cancer Center). ACK1, AR, EGFR, Her2, Her3, Mcl-1, Bcl-2, Puma, YB-1, FAK, AKT, Anti-phospho-AKT (Ser473), anti-phospho-S6K1 (S79), Anti-phospho-4-EBP1, Anti-phospho-LRP6, Anti-phospho-mTOR, Anti-phospho-YB-1, Anti-phospho-FAK (Y397), anti- ribosomal protein S6, anti-ribosomal protein S6, BCL-xL and anti-cleaved Casp3 antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-phospho-ribosomal protein S6 (S235/236), mouse anti-ERK antibody and Anti-phospho-ERK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Vinculin was purchased from Sigma. Secondary anti-mouse IgG with horseradish peroxidase was from Calbiochem. Secondary anti-rabbit IgG with horseradish peroxidase was from GE Healthcare. Ceritinib and Paclitaxel were purchased from APExBIO. Bicalutamide (97%) purchased from ACROS Organics. The 133 FDA-approved drugs were kindly provided from NCI/DTP Approved Oncology Drugs Plated Set (AODVIII).
Dong S., Yousefi H., Savage I.V., Okpechi S.C., Wright M.K., Matossian M.D., Collins-Burow B.M., Burow M.E, & Alahari S.K. (2022). Ceritinib is a novel triple negative breast cancer therapeutic agent. Molecular Cancer, 21, 138.
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8

Antibody Characterization for Cell Adhesion

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Antibodies were purchased: monoclonal antibodies, anti-integrin αvβ3, anti-integrin β3(CD61), and anti-integrin α5β1 (Millipore, Billerica, MA), anti-human CD171 (L1CAM; BD Pharmingen, San Diego, CA), anti-integrin αvβ5 (R&D Systems, Minneapolis, MN), anti-actin, anti-β-tubulin, anti-phospho-ERK, and anti-phospho-JNK (Santa Cruz, Dallas, TX), and anti-L1CAM UJ127 (GeneTex, Irvine, CA); rabbit antibodies, anti-integrin β3 (Epitomics, Burlingame, CA), and anti-phospho-FAK (Y397), anti-total FAK, anti-phospho-BMX/Etk (Y40), anti-total BMX/Etk, and anti-total p130CAS (E1L9H) (Cell Signaling, Beverly, MA). Rabbit affinity purified phospho-specific anti-p130CAS (Y234) was generated using the following peptide: -AQPEQDE[pY]DIPRHL, corresponding to residues 227-240 in human p130CAS, by 21st Century Biochemicals, Inc (Marlboro, MA) (SFigure 4).
Burgett M.E., Lathia J.D., Roth P., Nowacki A.S., Galileo D.S., Pugacheva E., Huang P., Vasanji A., Li M., Byzova T., Mikkelsen T., Bao S., Rich J.N., Weller M, & Gladson C.L. (2016). Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells. Oncotarget, 7(28), 43852-43867.
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9

Immunofluorescence and Western Blot Analysis of Rab13

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For IF, cells were plated onto collagen IV-coated glass coverslips (10µg/ml) and then fixed with 4% paraformaldehyde (PFA) in PBS (phosphate-buffered saline) for 15min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, blocked with 5% goat serum in PBS for 1h, and incubated with Rab13 rabbit polyclonal antibody (Novus Biologicals, cat# NBP1-85799, 1/200 dilution) for 1h. Secondary antibodies were conjugated with Alexa 647 (Thermo Fisher Scientific).
For Western blot detection the following antibodies were used: anti-Rab13 rabbit polyclonal (Novus Biologicals, cat# NBP1-85799, 1/1,000 dilution), anti-GFP rabbit polyclonal (Invitrogen, cat# A11122, 1/2,000 dilution), anti-RhoGDI, anti-Transferrin receptor (clone H68.4, ThermoFisher Scientific, cat# 13-6800), anti-a-tubulin mouse monoclonal (Sigma-Aldrich, cat# T6199, 1/10,000 dilution), and anti-GAPDH rabbit monoclonal 14C10 (Cell Signaling Technology, cat# 2118, 1/2,000 dilution), anti-phospho-FAK(Y397) (Cell Signaling Technology, cat# 3283), anti-REP-1 (Abcam, cat# 134964), anti-RabGDI (Santa Cruz Biotechnology, cat# sc-374649), anti-RABIF mouse monoclonal antibody D-12 (Santa Cruz biotechnology, cat# sc-390759, 1/1,000 dilution).
Moissoglu K., Stueland M., Gasparski A.N., Wang T., Jenkins L.M., Hastings M.L., & Mili S. (2020). Local RNA translation controls cell migration and Rab GTPase function.
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10

Antibody Procurement for FAK, Paxillin, and GAPDH

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Rabbit polyclonal anti-phospho-Y397-FAK, anti-FAK, anti-phospho-Y118-Paxillin, and anti-GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-integrin β1 antibodies were obtained from BD Biosciences and Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-Paxillin antibody was from BD Transduction Laboratories (San Jose, CA, USA). The secondary anti-mouse and anti-rabbit antibodies tagged with HRP were purchased from Promega (Madison, WI, USA). Rabbit polyclonal anti-claudin-7 antibody was obtained from Immuno-Biological Laboratories (Gunma, Japan), and mouse monoclonal anti-Myc antibody was obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).
Kim D.H., Lu Q, & Chen Y.H. (2019). Claudin-7 modulates cell-matrix adhesion that controls cell migration, invasion and attachment of human HCC827 lung cancer cells. Oncology Letters, 17(3), 2890-2896.
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