Immunoblotting was performed with rabbit anti‐ITGA3 antibody (1:250, HPA008572; SIGMA‐ALDRICH, St. Louis, MO, USA), anti‐Akt antibody (1:1000, #4691; Cell Signaling Technology, Danvers, MA, USA), anti‐p‐Akt antibody (1:1000, #4060; Cell Signaling Technology), anti‐Erk1/2 antibody (1:1000, #4695; Cell Signaling Technology) and anti‐p‐Erk1/2 antibody (1:2000, #4370; Cell Signaling Technology), anti‐FAK antibody (1:1000, #3285; Cell Signaling Technology) and anti‐p‐FAK antibody (1:1000, #8556; Cell Signaling Technology). Anti‐GAPDH antibodies (1:1000, ab8245; Abcam, Cambridge, UK) were used as an internal control. The procedures were performed as described in our previous studies.15 , 16 , 22
Anti fak antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-FAK Antibody is a highly specific and sensitive tool for the detection of Focal Adhesion Kinase (FAK) protein. FAK is a non-receptor protein tyrosine kinase that plays a crucial role in cell adhesion, migration, and signaling. This antibody can be used to identify and quantify FAK expression in various cell and tissue samples.
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Lab products found in correlation
Anti erk1 2 antibody, by Cell Signaling Technology (2 mentions)
Anti p erk1 2 antibody, by Cell Signaling Technology (2 mentions)
Anti p fak antibody, by Cell Signaling Technology (2 mentions)
Anti akt antibody, by Cell Signaling Technology (2 mentions)
Anti p akt antibody, by Cell Signaling Technology (2 mentions)
Hpa008572, by Merck Group (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Bicinchoninic acid kit, by Thermo Fisher Scientific (1 mentions)
Pierce protein a g agarose beads, by Thermo Fisher Scientific (1 mentions)
Normal rabbit igg, by Merck Group (1 mentions)
Anti mouse igg antibody, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor mix, by Merck Group (1 mentions)
Anti grb2, by Abcam (1 mentions)
Anti ilk 4g9, by Cell Signaling Technology (1 mentions)
Anti src antibody, by Cell Signaling Technology (1 mentions)
Anti p src antibody, by Cell Signaling Technology (1 mentions)
Sab2701430, by Merck Group (1 mentions)
Rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
13 protocols using anti fak antibody
1
Immunoblotting of Integrin, Akt, Erk, and FAK
2
Immunoprecipitation of Focal Adhesion Kinase
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HCT-116 cells were detached using trypsin-EDTA (TE) and washed twice with cold PBS. Cells were lysed using IP lysis buffer (Pierce, ThermoFisher Scientific, Waltham, MA, USA) and centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was collected and stored at −20 °C. A bicinchoninic acid kit (ThermoFisher Scientific) was used to measure protein concentrations. For IP, lysate containing 1500 µg total protein was mixed with either 10 µl anti-FAK antibody (Cell Signaling Technology, MA, USA) or normal rabbit IgG (Merck, NJ, USA) as a negative control in a total volume of 500 µl. Before IP, the lysates were precleared with 10 µl agarose slurry control to remove nonspecific binding between proteins and beads for 3 h at 4 °C with end-over-end rotation. Pierce™ Protein A/G Agarose beads (ThermoFisher Scientific) were preblocked with 1% BSA in PBS under the same conditions as those used for lysate preclearance. Then, antibodies were added to the clarified lysate for 2 h at RT with end-over-end rotation, followed by incubation with preblocked agarose slurry in PBS overnight at 4 °C. Beads were washed 5 times with 500 µl PBS-Tween 0.1% and 3 times with 500 µl PBS, resuspended in 40 µl 2X Laemmli Sample Buffer (Merck, NJ, USA) and boiled for 10 min at 95 °C. Next, proteins were separated on a 10% SDS-PAGE gel and visualized with an EzStain Silver kit (ATTO, Japan).
3
Western Blot Analysis of Focal Adhesion Kinase
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OS cells were collected, and western blotting was performed as previously described [13 (link)]. Blots were blocked in TBS with 0.05% Tween 20 (Sigma-Aldrich) buffer (TBS-T) containing 1% Bovine Serum Albumin (BSA) (013-27054, Fujifilm), followed by incubation in TBS-T with 1% BSA and primary antibodies. Primary antibodies used were anti-FAK antibody (#13009, 1:1000, Cell Signaling Technology, MA, USA), anti-phospho-FAK antibody (44-624G, 1:1000, Invitrogen, MA, USA), anti-CXCR1 antibody (BS60349, 1:500, Bioworld, Bloomington, MN, USA), anti-CXCR2 antibody (20634-1 AP, 1:1000, Proteintech, IL, USA), and anti-β-actin antibody (A2228, 1:1000, Sigma-Aldrich). After washing thrice with TBS-T, membranes were incubated with secondary antibodies. Secondary antibodies used were anti-rabbit IgG antibody (#7074, 1:2000, Cell Signaling Technology) and anti-mouse IgG antibody (#7076, 1: 2000, Cell Signaling Technology). Images were captured using a ChemiDoc Touch and quantified using Image J software.
4
Immunoprecipitation of Integrin-Linked Kinase
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β1WT and β1KO Fo and transitional B cells were lysed with buffer containing 20 mM Hepes (pH 7.6), 2 mM MgCl2, 150 mM NaCl, 10% glycerol, 0.1% NP40, 1 mM PMSF, and protease inhibitor mix (Sigma-Aldrich). Following sonication, the cell lysate was centrifuged at 16,100 g for 10 min at 4°C. The protein concentration of the supernatant was measured by Bradford assay, and 1 mg of total protein was then mixed with anti-ILK (4G9; rabbit monoclonal; Cell Signaling Technology) or anti-rabbit IgG isotype control antibody (Biomol) and rotated ON at 4°C. Subsequently, samples were incubated with Dynabeads Protein G (Thermos Fischer Scientific) for 2 h at 4°C, beads were washed, and associated proteins were eluted by addition of 2× sample buffer and a boiling step of 10 min at 95°C. Following separation of the proteins by SDS-PAGE, proteins were detected with anti-ILK (4G9; rabbit monoclonal; Cell Signaling Technology), anti-Grb2 (Y237; rabbit monoclonal; Abcam), and anti-FAK antibody (rabbit polyclonal; Cell Signaling Technology).
5
Immunoblotting Analysis of Key Signaling Proteins
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Cells were harvested 48 h after transfection, and lysates were prepared. Immunoblotting was carried out with rabbit anti‐SKA1 antibodies (1:1,000 dilution, SAB2701430; Sigma‐Aldrich), anti‐AKT antibody (1:1000, #4691; Cell Signaling Technology, Danvers, MA, USA), anti‐p‐AKT antibody (1:1000, #4060; Cell Signaling Technology), anti‐ERK1/2 antibody (1:1000, #4695; Cell Signaling Technology), anti‐p‐ERK1/2 antibody (1:2000, #4370; Cell Signaling Technology), anti‐FAK antibody (1:1000, #3285; Cell Signaling Technology) and anti‐p‐FAK antibody (1:1000, #8556; Cell Signaling Technology), anti‐SRC antibody (1:1000, #2123; Cell Signaling Technology) and anti‐p‐SRC antibody (1:1000, #6943; Cell Signaling Technology). Anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibodies (1:10000, ab8245; Abcam, Cambridge, UK) were used as an internal loading control. Experimental procedures were carried out as described previously.22 , 25 , 26 , 27 , 28 Protein expression was quantified by using NIH‐ImageJ.
6
RNA-Binding Protein Immunoprecipitation of circCDYL2
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The RIP assay was performed according to RNA-Binding Protein Immunoprecipitation Kit manual (17–700, Millipore, USA). In brief, SK-BR-3 cells were washed by cool PBS twice and lysed by co-IP buffer. The lysates were first co-incubated with anti-GRB7 antibody (376,069, Santa Cruz, USA, 1:1000), or negative control IgG (2,822,115, Millipore, USA, 1:1000) and anti-FAK antibody (13,009, Cell Signaling Technology, USA, 1:1000) overnight at 4 °C. Subsequently, the protein A/G beads were co-incubated with immunocomplex mixture for 2 h in 4 °C. Total RNA of the immunocomplex was extracted by TRIzol reagent (Invitrogen, Carlsbad, USA), then the circCDYL2 expression was detected by qRT-PCR.
7
Co-Immunoprecipitation of GRB7 and FAK
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The Co-IP assay was performed according to previous literature [28 (link)]. SK-BR-3 cells were washed by cool PBS twice and lysed by co-IP buffer. The lysates were first co-incubated with anti-GRB7 antibody (376,069, Santa Cruz, USA, 1:1000) or anti-FAK antibody (13,009, Cell Signaling Technology, USA, 1:1000) overnight at 4 °C. Subsequently, the protein A/G beads were co-incubated with mixture for 2 h in 4 °C. The beads were washed with PBS, then resuspended by 1 × SDS-PAGE Loading Buffer. Total protein of the immunocomplex was subjected to Western Blot with antibodies against the other protein. For detecting the ubiquitination of GRB7, cells were treated with MG132 (474790, Sigma-Aldrich, USA).
8
Immunoprecipitation of Integrin β1
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The post-infection cell monolayers were washed twice with ice-cold PBS and lysed in 200 μl IP buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) Na-deoxycholate, 1 mM EDTA, proteinase inhibitor cocktail) for 15 min on ice. The resulting cell lysates were transferred into pre-cooled 1.5 ml tubes, passed through a 29-gauge needle twice, and centrifuged for 10 min at 14,000 g, 4°C. An aliquot of the supernatant was sampled for input protein content analysis. The remaining supernatants were pre-cleared with Protein G agarose beads (Thermo Scientific) with rotation for 30 min at 4°C. The pre-cleared supernatants were incubated with anti-integrin β1 antibody (rat monoclonal IgG1, 1:100, DSHB) overnight with rotation at 4°C. Then the Protein G magnetic agarose was added into the tubes and co-incubated overnight at 4°C with rotation. The next day, tubes were placed on the magnetic stand to collect beads. The beads were washed with IP buffer 5 times, then resuspended in 100 μl of loading buffer and heated to 100°C for 10 min to elute proteins. The supernatants collected were used for immunoblotting with anti-intimin-γ antibody (Gift from Dr. John M Leong) or anti-FAK antibody (Cell signaling), respectively.
9
FAK Expression in PDAC Vasculature
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Human pancreatic cancer samples (Sun Yat-Sen memorial hospital) were obtained with signed informed consent from patients and ethical committee approval. Patients with PDAC all received gemcitabine-based treatment after surgery and their clinical responses to gemcitabine were evaluated and recorded (n = 40 patients), and some of these patients with their relapse information (n = 26 patients with PDAC). FFPE human PDAC tissues were cut into 4-mm sections and subjected to immunofluorescent staining as previously described (30 (link)). Antibodies used for immunofluorescent staining were mouse monoclonal anti-CD34 antibody against human cell surface glycoprotein (ZSGB-BIO, ZM-0046, 1:100 dilution) and rabbit monoclonal anti-FAK antibody against human or mouse (Cell Signaling Technology, 3285S, 1:100 dilution). For the immunofluorescence analysis, Alexa Fluor–conjugated secondary antibodies (Invitrogen Molecular Probes) were used to detect human antigens, and the sections were counterstained with DAPI (Sigma-Aldrich, D8417). For data analysis, the percentage of FAK negative blood vessels was calculated as the number of CD34-positive blood vessels that were negative for FAK over the total number of CD34-positive blood vessels. Patient data are expressed as those with either less, or more, than 44% of blood vessels that are FAK negative.
10
Ovarian Cancer Cell Line Cultivation
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The human OC cell lines CaOV3 and OVCAR3 were obtained from the American Type Culture Collection (Manassas, VA) and maintained in a humidified 5 % CO2 incubator at 37 °C. Cells were passaged twice weekly. OVCAR3 cells were maintained in RPMI-1640 (Wisent, St-Bruno, QC, Canada) supplemented with 20 % FBS, insulin (10 mg/L), glutamine (2 mM) and antibiotics. CaOV3 cells were cultured in DMEM/F12 (Wisent) supplemented with 10 % FBS, 2 mM glutamine and antibiotics. Acellular ascites fractions were obtained at the time of initial cytoreductive surgery from women with advanced serous ovarian carcinomas. Samples were supplied by the Banque de tissus et de données cliniques et biologiques sur les cancers gynécologiques et du sein de Sherbrooke as part of the Banque de tissus et de données du Réseau de Recherche en Cancer des Fonds de Recherche du Québec en Santé (FRQS) affiliated to the Canadian Tumor Repository Network (CTRNet). HRP-conjugated anti-mouse and rabbit antibodies and anti-FAK antibody were purchased from Cell Signaling Technology (Danvers, MA). Anti-phospho FAK and anti-phospho Pyl2 were from Thermo Fisher (Waltham, MA). Anti-Pyk2 and anti-Tubulin were purchased from Sigma-Aldrich (Oakville, ON). CCL18 and CCL18 neutralizing antibody were from RnD Systems (Minneapolis, MN). Plasmid pCMV6-ENTRY-PTK2B was obtained from Origene (Rockville, MD).
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