The transfected cells and tumor tissues were lysed in RIPA lysis buffer (CWBio) on ice for 30 min and the mixture was centrifuged for 15 min (4°C, 14,000 × g) to obtain the supernatant. Following quantitative analysis using a BCA kit (Beyotime Institute of Biotechnology), the proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gels by electrophoresis, transferred to polyvinylidene difluoride membranes and incubated with 5% non-fat milk for 2 h at room temperature. Subsequently, the membranes were incubated with antibodies against matrix metalloproteinase (MMP)2 (ab92536; dilution, 1:1,000), MMP9 (ab76003; dilution, 1:1,000), Ki67 (ab92742; dilution, 1:5,000), survivin (ab208938; dilution, 1:1,000), β-catenin (ab6302; dilution, 1:4,000), c-MYC (ab32072; dilution, 1:1,000), cyclin D1 (ab16663; dilution, 1:200), phosphorylated (p)-FAK (ab81298; dilution, 1:1,000), p-AKT (ab81298; dilution, 1:1,000) and GAPDH (ab9485; 1:1,000) (all from Abcam) at 4°C overnight. The following day, the membranes were incubated with a horseradish peroxidase-labeled secondary antibody (#7074; 1:1,000; Cell Signaling Technology, Inc.) at room temperature for 1 h and visualized using ECL reagents (Research-bio). Relative protein expression was quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.).
Ab81298
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab81298 is a polyclonal antibody product designed for use in various research applications. As a laboratory reagent, its core function is to specifically detect and bind to the target antigen. No further details on intended use are provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab40794, by Abcam (11 mentions)
Ab76003, by Abcam (3 mentions)
Ab92536, by Abcam (3 mentions)
Ab131435, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab81283, by Abcam (3 mentions)
Ab32084, by Abcam (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Mab374, by Merck Group (2 mentions)
Ab205718, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Ab150077, by Abcam (2 mentions)
Ab129002, by Abcam (2 mentions)
Hpa007415, by Merck Group (2 mentions)
Sc 558, by Santa Cruz Biotechnology (2 mentions)
Ab21685, by Abcam (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Ethidium homodimer 1, by Thermo Fisher Scientific (2 mentions)
31 protocols using ab81298
1
Protein Expression Analysis in Transfected Cells
2
Immunoblotting Analysis of Focal Adhesion Kinase Signaling
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The reagents used were as follows: rabbit monoclonal antibodies against p-FAK (Tyr397) (1:1000, ab81298, Abcam), flag(1:1000, 14793 S, Cell Signaling Technology), myc (1:1000, 2278t, Cell Signaling Technology), SRF (1:500, #5147, Cell Signaling Technology), N-Cadherin (1:1000, #13116, Cell Signaling Technology), NgR3 (1:1000, NBP1-92359, Novus); rabbit polyclonal antibodies against FAK (1:1000, #3285 S, Cell Signaling Technology dilution); p-SRF (Ser103) (1:500, #4261, Cell Signaling Technology); Alexa Fluor® 594 (or 488)-conjugated goat-anti-mouse (or rabbit) (1:2000, ThermoFisher); mouse monoclonal antibodies against GAPDH (1:2000, KC-5G4, Kangcheng Biotech). Nuclear dye against 40–60-diamidino-2-phenylindole (DAPI; 1:2000, Molecular Probes).
3
Mechanosensitive Protein Expression in mPFBs
4
Western Blot Analysis of Signaling Proteins
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Cell lysates were separated by SDS-PAGE, followed by wet transfer onto 0.45 µm PVDF membranes, which were then blocked with 5% BSA in PBST solution for 30 min. Specific primary antibody incubation was conducted overnight at 4 °C. The antibodies used were as follows: anti-phospho-Y397-Fak (ab81298, Abcam, USA), anti-FAK (ab131435, Abcam, USA), anti-Tyr416-phosphor-c-Src (#2010s, CST), anti-c-Src (#2108s, CST), anti-Ser473-phospho-Akt (#4060s, CST), anti-Akt (#9272, CST, Danvers, MA), anti-FASN (#14979-1-AP, Proteintech, Rosemont, USA), anti-β-actin (#sc-81178, Santa Cruz, USA). Secondary antibody was incubated for 1 h at RT. Immunodetection was accomplished using enhanced chemiluminescence. The band intensity was determined with ImageJ software. All the original western blot pictures were provided in the “Supplemental Material original western blots”.
5
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Cells were treated with radioimmunoprecipitation assay buffer (RIPA, Beyotime, China) supplemented with 1% protease and phosphatase inhibitors (CWBIO, China). Equal amounts of protein were electrophoresed and transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked by 5% nonfat milk in TBST buffer for one hour and then incubated with primary antibodies against the following proteins: DHX9 (ab26271, Abcam, UK, RRID:AB_777725 ), GAPDH (AC002, ABclonal, China, RRID:AB_2736879 ), STEAP4 (ab63967, Abcam, UK, RRID:AB_1143135 ), FAK (ab40794, Abcam, UK, RRID:AB_ 732,300), pFAK (phospho Y397) (ab81298, Abcam, UK, RRID:AB_1640500 ), ZO-1 (#8193, CST, US, RRID:AB_10898025 ), E-Cadherin (#3195, CST, US, RRID:AB_2291471 ), Vimentin (#5741, CST, US, RRID:AB_10695459 ), N-Cadherin (#13116, CST, US, RRID:AB_2687616 ), ZEB1 (#3396, CST, US, RRID:AB_1904164 ), Slug (#9585, CST, US, RRID:AB_2239535 ), and β-Catenin (#9562, CST, US, RRID:AB_331149 ). The next day, the membranes were washed with TBST buffer and incubated with HRP-conjugated secondary antibodies. Signal intensities were measured by Immobilon ECL substrate (Millipore, Germany), and images were acquired using an OPTIMAX X-ray Film Processor (Protec, Germany).
6
Protein Expression Analysis in Tumor Samples
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Proteins extracted from tumor tissue and cells were separated by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred to Polyvinylidene Fluoride (PVDF) membranes (Sigma Aldrich). Following blockade with bovine serum albumin (Sigma Aldrich) and incubation with primary antibodies against DSG3 (1:2000, ab218380, Abcam), Src (ab47405) and p-Src (1:2500, p-Tyr418, ab4816 Abcam), FAK (ab131435) and p-FAK (1:3000, p-Tyr397, ab81298, Abcam), AKT (ab235958) and p-AKT (1:3500, p-Ser 473, ab81283, Abcam), and β-actin (1:4000, ab8227, Abcam), the membrane was then incubated with HRP-conjugated secondary antibody (1:5000, ab205718, Abcam), and the signals were detected with chemiluminescence system (Tanon, Shanghai, China).
7
Immunostaining of Cytoskeletal Markers
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CFs were penetrated and incubated with the following primary antibodies at 4 °C overnight: anti-alpha smooth muscle actin antibody (α-SMA, 1:800 dilution, Abcam, ab7817, Cambridge, UK), anti-focal adhesion kinase (phospho Y397) antibody (FAK, 1:800 dilution, Abcam, ab81298, Cambridge, UK), and anti-angiotensin II type-1 receptor antibody (AT1R, 1:100 dilution, Proteintech, 25343-1-AP, Chicago, IL, USA). Then, the cell samples were incubated with the following secondary antibodies in the dark at 37 °C for 2 h: Alexa Fluor-488 goat anti-mouse antibody (1:1000 dilution, Abcam, Ab150077, Cambridge, UK) or Alexa Fluor-594 goat anti-rabbit antibody (1:1000 dilution, Abcam, Ab150116, Cambridge, UK). Cell nuclei were stained using DAPI (1 μg mL−1, Sigma, D9542, St. Louis, MO, USA). Images of the cell samples were obtained using a laser scanning confocal microscope (FV3000 Olympus, Tokyo, Japan).
8
Quantifying FA Protein Expression
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The expression levels of FA proteins were quantified using western blotting (WB) at room temperature (RT). Whole cell proteins were extracted using NuPAGE™ LDS Sample Buffer (Thermo Fisher, Waltham, MA, USA), loaded on Bolt™ Bis-Tris Protein Gel (Thermo Fisher), and blotted onto 0.45 μm PVDF membranes (Thermo Fisher). Proteins were quantified using a Qubit™ Protein Broad Range Assay and Qubit 4 Fluorometer (Thermo Fisher) following the manufacturer’s instruction. Membranes were finally incubated with primary antibodies: GAPDH (AM4300, Thermo Fisher), vinculin (ab129002, abcam), paxillin (ab32084, abcam), FAK (ab40794, abcam), and phospho Y397 FAK (ab81298, abcam) and secondary antibodies: Goat Anti-Rabbit IgG H&L HRP (ab205718, abcam) and Goat Anti-Mouse IgG H&L HRP (G21040, Thermo Fisher) using iBind Western System (Thermo Fisher). All membranes were imaged using a ChemiDoc Touch MP system (Bio-Rad Laboratories, Hercules, CA, USA). Bands were quantified using ImageJ, and statistical data were analyzed using GraphPad Prism 9.
9
Immunofluorescence Analysis of Focal Adhesion Kinase
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HeLa, CaSki, and C33A cells were seeded on coverslips and exposed to treatments. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton X for 5 min. Blocking was performed with 5% bovine serum albumin (BSA) for 20 min. Cells were incubated with primary antibodies against phospho-Y397-Fak (ab81298, Abcam, USA) overnight at 4 °C and then with FITC-conjugated secondary antibody at RT for 1 h. After washing, F-actin was stained with Acti-stain 555 phalloidin (#PHDH1, Cytoskeleton, Inc.) The nuclei were counterstained with 4′-6-diamidino-2-phenylindole (Sigma-Aldrich). The coverslips were mounted with Vectashield mounting medium (Vector Laboratories, CA). Immunofluorescence was visualized using an Olympus BX41 microscope.
10
Quantifying Focal Adhesions by Immunofluorescence
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Immunofluorescent staining was performed to visualize FA proteins: vinculin, paxillin, and FAK. As mentioned in Section 2.1.4 , cells on substrates were fixed with 4% PFA, permeabilized with 0.5% Triton X-100, and blocked with Blocking One Histo. Substrates were then incubated with primary antibodies overnight at 4 °C: vinculin (ab129002, abcam, Cambridge, UK), paxillin (ab32084, abcam), and phospho Y397 FAK (ab81298, abcam) following dilution protocols before labeling with secondary antibodies: Goat Anti-Rabbit IgG H&L Alexa Fluor® 488 (ab150077, abcam) or Goat Anti-Rabbit IgG H&L Alexa Fluor® 647 (ab150083, abcam) for 30 min following dilution protocols. Actin filament and nuclei were stained for 30 min using Acti-stain™ 555 Fluorescent Phalloidin and DAPI, then substrates were imaged using a fluorescence microscope BX-53 with a UPLX Apo 10× objective lens or BZ-X710 microscope (Keyence, Osaka, Japan) with a Plan Apo Lambda 10× or 20× objective lens. Using a protocol designed to quantify the FAs [42 (link), 43 (link)], the average size of the FAs, number of FAs in a cell adhesion site, cell adhesion site, and FA density were quantified using ImageJ software.
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