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Total fak

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Total FAK is a protein quantification assay that measures the total amount of Focal Adhesion Kinase (FAK) in a sample. FAK is a cytoplasmic protein-tyrosine kinase that plays a key role in the regulation of cell growth, survival, migration, and differentiation. The Total FAK assay provides a quantitative assessment of FAK levels in various biological samples.

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Lab products found in correlation

P fak, by Cell Signaling Technology (4 mentions) Total akt, by Cell Signaling Technology (3 mentions) Total erk, by Cell Signaling Technology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) P akt, by Cell Signaling Technology (2 mentions) P stat5, by Cell Signaling Technology (1 mentions) Total stat5, by Cell Signaling Technology (1 mentions) β tubulin, by Cell Signaling Technology (1 mentions) P fak y397, by Cell Signaling Technology (1 mentions) Nanog, by Cell Signaling Technology (1 mentions) Pierce ecl plus, by Thermo Fisher Scientific (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Radio immunoprecipitation assay ripa buffer, by Beyotime (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Phosphor erk, by Cell Signaling Technology (1 mentions) Phosphor fak, by Cell Signaling Technology (1 mentions) Erk1 2, by Cell Signaling Technology (1 mentions) P erk1 2, by Cell Signaling Technology (1 mentions) Enhanced chemiluminescence, by Cytiva (1 mentions) Pfak tyr 925, by Cell Signaling Technology (1 mentions)

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Anti-Total FAK (2 citations)

13 protocols using total fak

1

Western Blot Analysis of Signaling Proteins

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Cells were lysed in RIPA buffer containing protease inhibitors and protein lysates were subjected to SDS–PAGE and immunoblot analysis as previously described [34 (link)]. Antibodies used include pSTAT5 (Cell Signaling #9314, 1:1000), total STAT5 (Cell Signaling #9363, 1:1000), β-tubulin (Cell Signaling # 2146S, 1:1000), pFAK Y397 (Cell Signaling, # 3283S, 1:1000), and total FAK (Cell Signaling #3285S, 1:1000).
Jesser E.A., Brady N.J., Huggins D.N., Witschen P.M., O’Connor C.H, & Schwertfeger K.L. (2021). STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment. Breast Cancer Research : BCR, 23, 104.
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2

Western Blot Analysis of Cellular Proteins

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Cell lysates (20 μg total protein) as described34 (link) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to PVDF membrane. After blocking, membranes were incubated overnight at 4  °C with primary antibodies against Cx26 (Invitrogen, Grand Island, NY, USA), Cx43 (Cell Signaling, Danvers, MA, USA), NANOG (Cell Signaling, Danvers, MA, USA), GFP (Invitrogen, Grand Island, NY, USA), SOX2 (Cell Signaling, Danvers, MA, USA), OCT4 (Cell Signaling, Danvers, MA, USA), phospho-FAK (Y397, Y576, Y925) (Cell Signaling, Danvers, MA, USA), total FAK (Cell Signaling, Danvers, MA, USA), and/or β-actin (Santa Cruz, Dallas, TX, USA), followed by incubation with secondary anti-mouse or anti-rabbit immunoglobulin G (IgG) antibodies conjugated to horseradish peroxidase (Thermo Scientific, Waltham, MA, USA). Immunoreactive bands were visualized by exposing films to luminescent signals generated after incubating the membrane with Pierce ECL plus (Thermo Scientific, Waltham, MA, USA). Copies of the uncropped blot scans are provided in the supplementary information (Supplementary Figs. 14–38) with associated molecular weights indicated for each antibody.
Thiagarajan P.S., Sinyuk M., Turaga S.M., Mulkearns-Hubert E.E., Hale J.S., Rao V., Demelash A., Saygin C., China A., Alban T.J., Hitomi M., Torre-Healy L.A., Alvarado A.G., Jarrar A., Wiechert A., Adorno-Cruz V., Fox P.L., Calhoun B.C., Guan J.L., Liu H., Reizes O, & Lathia J.D. (2018). Cx26 drives self-renewal in triple-negative breast cancer via interaction with NANOG and focal adhesion kinase. Nature Communications, 9, 578.
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3

Protein Extraction and Western Blot Analysis

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Total protein samples were extracted by lysing cells with an equivalent Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, Haimen, China). The total cellular protein samples were separated by electrophoresis on SDS-PAGE gels and transferred to a polyvinylidene difluoride (PVDF) membrane (Biorad). The membrane was blocked with 5% nonfat milk or BSA in TBST buffer at room temperature for 2 h, and incubated with primary antibodies raised against phosphor-Erk, total-Erk, phosphor-FAK, total-FAK, and GAPDH (Cell Signaling Technology, Inc., Boston, MA, USA) at 4 °C, with sufficient contact overnight. Then the membrane was incubated with secondary antibodies. The blots were measured and visualized by enhanced chemiluminescence (ECL) reagent, followed by ImageJ software quantification and normalization.
Liu Y., Ding Y., Ye M., Zhu T., Tian D, & Ding K. (2017). A Novel Heterogalactan from Antrodia camphorata and Anti-Angiogenic Activity of Its Sulfated Derivative. Polymers, 9(6), 228.
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4

Western Blot Analysis of Signaling Proteins

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Western blot analysis was performed, as described previously [44 (link)]. Briefly, whole-cell lysates were prepared in a modified RIPA buffer containing proteinase inhibitors and phosphatase inhibitors as described elsewhere [45 (link)]. Equivalent amounts of protein (20–80 μg) were loaded in 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred by blotting to polyvinylidene fluoride (PVDF) membranes. The blot was incubated with primary antibodies against pFAK (Tyr 925; Cell Signaling Technology), pFAK (Tyr 576/577: Cell signaling technology), total FAK, pAKT, AKT, pERK1/2, ERK1/2 (Cell Signaling Technology), and actin (Santa Cruz Biotechnology, Inc.). After washing, the blot was incubated with HRP-conjugated secondary antibodies. The protein–antibody complexes were detected using enhanced chemiluminescence (Amersham, Arlington Heights, IL, USA), according to the manufacturer's recommended protocol.
Western blot bands were quantified using Image J software (NIH, Bethesda, MD, USA). The adjusted relative densities were calculated relative to the expression of control sample's actin
Woo J.K., Jang Y.S., Kang J.H., Hwang J.I., Seong J.K., Lee S.J., Jeon S., Oh G.T., Lee H.Y, & Oh S.H. (2016). Ninjurin1 inhibits colitis-mediated colon cancer development and growth by suppression of macrophage infiltration through repression of FAK signaling. Oncotarget, 7(20), 29592-29604.
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5

Inhibitor-based Signaling Pathway Analysis

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Small-molecule FAK inhibitor PND-1186 and TAK1 inhibitor Takinib were purchased from Selleck Chemicals (Houston, TX). LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, total p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK, p-FAK, total FAK, p-TAK1, total TAK1, inhibitor of κBα (IκBα), p65 subunit of NFκB, p-p65, IκB kinase β (IKKβ), and p-IKKα/β were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against GAPDH and F4/80 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lamin B antibody was purchased from Abcam (Abcam, USA). Secondary antibodies were obtained from Santa Cruz Biotechnology.
Chen X., Zhao Y., Wang X., Lin Y., Zhao W., Wu D., Pan J., Luo W., Wang Y, & Liang G. (2022). FAK mediates LPS-induced inflammatory lung injury through interacting TAK1 and activating TAK1-NFκB pathway. Cell Death & Disease, 13(7), 589.
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6

Signaling Pathway Characterization in Cancer

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Recombinant Human Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF) were purchased from Peprotech. SGX523 was purchased from GE and Erlotinib from Cayman chemicals; both were solubilised in DMSO. All antibodies (Abs) were purchased from commercial sources as indicated. For tissue and cell staining, Phospho-Histone H3 was from Millipore, α-SMA from AnaSpec Inc., cd31 from Abcam, total cMET (D1C2) from Cell Signaling Technology and EGFR Ab from Novocastra/Leica. For immunoprecipitation, the EGFR Ab was from Santa Cruz Biotechnology (sc-120). For Western Blot, phospho-EGFR (Y1173), total EGFR phospho-MET (Y1234), total MET, phospho-ERK (Thr202/Tyr204), total ERK, phopho-AKT (Ser473), total AKT, phosphor-FAK (Tyr397) and total FAK were from Cell Signaling Technology; tubulin was from Millipore and hsc70 from Santa Cruz Biotechnology. For proliferation assays, anti-Brdu was purchased from Abcam.
Ortiz-Zapater E., Lee R.W., Owen W., Weitsman G., Fruhwirth G., Dunn R.G., Neat M.J., McCaughan F., Parker P., Ng T, & Santis G. (2017). MET-EGFR dimerization in lung adenocarcinoma is dependent on EGFR mtations and altered by MET kinase inhibition. PLoS ONE, 12(1), e0170798.
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7

Western Blot Analysis of DDR2 and FAK

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Total cell lysates were collected in Tris-Glycine SDS sample buffer (Invitrogen, LC2676) and fractioned using Tris-Glycine polyacrylamide gels (Invitrogen, XP04125). The following primary antibodies were used at a 1:1000 dillution: phosphorylated DDR2 at Y740 (B&D system, MAB25382), total DDR2 (LifespanBio, LS-B15752), phospho-FAK at Y397 (Cell Signaling, 3283) and total FAK (Cell Signaling 3285), were used at 1:1,000 dilution. Secondary antibody (horseradish peroxidase–conjugated goat anti-rabbit IgG) was used at 1:10,000 dilution and blots were visualized using ECL substrate (Amersham). Chemiluminescence signals were detected using a ChemiDoc Imaging system (Bio-Rad, ChemiDoc MP) and analyzed using Bio-Rad software Image Lab 6.0.1.
Ge C., Li Y., Wu F., Ma P, & Franceschi R.T. (2023). Synthetic Peptides Activating Discoidin Domain Receptor 2 and Collagen-Binding Integrins Cooperate to Stimulate Osteoblast Differentiation of Skeletal Progenitor Cells. Acta biomaterialia, 166, 109-118.
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8

Western Blotting Analysis of Protein Signaling

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Total protein lysates (20 µg) were resolved on a 4–12% bis-tris gel and transferred to Immobilon PVDF transfer membranes. Membranes were incubated for 40 min at room temperature in blocking solution (TRIS-buffered saline containing 5% nonfat dried milk and 0.1% Tween 20), followed by an overnight incubation in primary antibodies at 4°C. Membranes were then washed 3 times and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 h. After 3 additional washes, the immune complexes on the membranes were visualized by ECL detection.
The following antibodies were purchased and utilized in our study: Cell Signaling (Danvers, MA): phospho-AKT (#4058), total AKT (#2920), AKT2 (#3063), phospho-ERK 1/2 (#4695), total ERK ½ (#9107), PTEN (#9559), phospho-mTOR (#2971), total mTOR (#2972), phospho-MET (#3129 for western blotting and #3077 for IHC), c-MET (#3148), CD44 (#3570), phospho-Beta catenin (#4176), phospho-FAK (#8556), and total FAK (#3285). Santa Cruz (Dallas, TX): AKT 1 (#5298). BD (San Jose, California): Beta catenin (#610154) and E-cadherin (#610404). Abcam (Cambridge, MA): KRAS (#55391). Millipore (Billerica, MA): p85α (#05-212). All antibodies were used at a concentration of 1∶1,000.
Elliott V.A., Rychahou P., Zaytseva Y.Y, & Evers B.M. (2014). Activation of c-Met and Upregulation of CD44 Expression Are Associated with the Metastatic Phenotype in the Colorectal Cancer Liver Metastasis Model. PLoS ONE, 9(5), e97432.
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9

Estradiol and FAK Inhibitor Signaling

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Estradiol and FAK inhibitor (PF573228) were purchased from Sigma-Aldrich (St. Louis, MO); ICI 182,780 (ICI) was purchased from Tocris (Park Ellisville, MO). SERMs: 4-hydroxytamoxifen (4-OHT) was purchased from Sigma-Aldrich (St. Louis, MO), raloxifene was a kind gift from Eli Lilly (Indianapolis, IN), endoxifen was gifted from Dr. James Ingle (Mayo Clinic, Rochester, MN), bazedoxifene (BZA) was gifted from Dr. Ronald Grigg (University of Leeds, UK), EM652 was gifted from AstraZeneca (UK). c-Src inhibitor, PP2, and IGF-1Rβ inhibitor, AG1024, were purchased from CalBiochem (San Diego, CA). Sources of antibodies for Western blot were as follows: ERα (sc-544), mouse IGF-1Rβ (sc-462), and rabbit IGF-1Rβ (sc-713) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Total MAPK (#9102), phosphorylated MAPK (#9101), total Akt (#9272), phosphorylated Akt (#9271), total STAT3 (#4903), phosphorylated STAT3 (#9131), total FAK (#3285), phosphorylated FAK (Y397, #3283), phosphorylated FAK (Y576/577, #3281), phosphorylated p130CAS (#4014), phosphorylated IGF-1Rβ (#3024), and phosphorylated c-Src (#2101) antibodies were from Cell Signaling Technology (Beverly, MA). Total c-Src (GD11) and anti-phosphotyrosine 4G10 antibodies were from Millipore (Temecula, CA). Total p130CAS (Cat: 610272) was purchased from BD Biosciences (San Jose, CA).
Fan P., Agboke F.A., Cunliffe H.E., Ramos P, & Jordan V.C. (2014). A molecular model for the mechanism of acquired tamoxifen resistance in breast cancer. European journal of cancer (Oxford, England : 1990), 50(16), 2866-2876.
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10

Fibroblast Protein Expression Analysis

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At the end of the experiments, fibroblasts were harvested and whole cell lysates (100 μg) were subjected to Western-blot analysis25 (link) using antibodies for Type I collagen (Southern Biotech), α-SMA (Sigma), phospho-Smad2/3 (Cell-Signaling), phospho-FAK (pY397, Cell Signaling), total-FAK (Cell Signaling) or GAPDH (Zymed, San Francisco, CA). Electrophoretic bands were detected using enhanced chemiluminescence reagents (Pierce Biotechnology, Rockford, IL), and band intensities were quantified with Image J software. Results were normalized with GAPDH levels in each sample.
Marangoni R.G., Masui Y., Fang F., Korman B., Lord G., Lee J., Lakota K., Wei J., Scherer P.E., Otvos L., Yamauchi T., Kubota N., Kadowaki T., Asano Y., Sato S., Tourtellotte W.G, & Varga J. (2017). Adiponectin is an endogenous anti-fibrotic mediator and therapeutic target. Scientific Reports, 7, 4397.
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