mPFBs were cultured on plates of different elastic modulus (8 kPa, 0.2 kPa) coated with 100 µg/ml collagen type I (PureCol, Advanced BioMatrix, Carlsbad, CA) in DPBS for 24 h. Cell lysates were harvested with RIPA buffer (Sigma Aldrich) containing protease and phosphatase inhibitor cocktails. Protein concentration was determined by Pierce® BCA Protein assay kit (Pierce; Waltham, MA). 12 µg proteins of each sample were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were washed with Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and blocked with 5% dried milk in TBS-T or 5% bovine serum albumin (BSA) in TBS-T. Primary antibodies for fibronectin (ab1954, 1:2000, Millipore), α-SMA (A5228, 1:2000, Sigma Aldrich), pFAK Y397 (ab81298, 1:1000, Abcam), FAK (ab40794, 1:1000, Abcam), Vinculin (MAB3574, 1:1000, Millipore), β1-integrin (SAB5600100, 1:1000, Sigma-Aldrich), GAPDH (MAB374, 1:1000, Millipore) were used to incubate the membranes for 12 h. Detection was with appropriate peroxidase-conjugated secondary antibodies (1:2500, Jackson ImmunoResearch; West Grove, PA), which were developed with Clarity Western ECL substrate (Bio-Rad; Hercules, CA). Densitometry analysis was performed using Image Lab Software (Bio-Rad).
Ab40794
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab40794 is an antibody product manufactured by Abcam. It is a research-use only product designed for laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab81298, by Abcam (11 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Ab187027, by Abcam (4 mentions)
Ab32084, by Abcam (3 mentions)
Ab9485, by Abcam (3 mentions)
Ab125025, by Abcam (3 mentions)
Ab8227, by Abcam (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
A5228, by Merck Group (2 mentions)
Ab21685, by Abcam (2 mentions)
Ab92536, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab38449, by Abcam (2 mentions)
Ab92494, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
Ripa buffer, by Merck Group (1 mentions)
Mab374, by Merck Group (1 mentions)
Mab3574, by Merck Group (1 mentions)
27 protocols using ab40794
1
Mechanosensitive Protein Expression in mPFBs
2
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).
3
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.
4
Western Blot Profiling of Cellular Signaling
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Western blotting was carried out using an SDS-PAGE Electrophoresis System. Adherent cells or adipose tissue extracts were prepared and transferred to PVDF membranes. The primary antibodies for this experiment were as follows: anti-GAPDH (AB2000, Abways, Shanghai, China), anti-Col XV (ab202554, Abcam, Shanghai, China), anti-CHOP (YM3668, Immuno Way, Suzhou, China), anti-GRP78 (ab21685, Abcam, Shanghai, China), anti-IRE1α (ab124945, Abcam, Shanghai, China), anti-IL-6 (ab100712, Abcam, Shanghai, China), anti-MCP1 (ab100712, Abcam, Shanghai, China), anti-IL-1β (ab100712, Abcam, Shanghai, China), anti-CD206 (ab125028, Abcam, Shanghai, China), anti-CD163 (YM6146, Immuno Way, Suzhou, China), anti-TNFα (11948P, Cell signaling, Massachusetts, USA), anti-pFAK (ab81298, Abcam, Shanghai, China), anti-FAK (ab40794, Abcam, Shanghai, China), anti-Integrin β1 (ab24693, Abcam, Shanghai, China), Horseradish peroxidase anti-rabbit (Sigma-Aldrich, Shanghai, China) or anti-goat (Sigma-Aldrich, Shanghai, China) was used as secondary antibody. See Antibody Information Sheet (Appendix A ) for antibody details.
5
Western Blot Analysis of Protein Markers
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Proteins were extracted with radioimmunoprecipitation buffer containing proteinase inhibitor (Beyotime), and then separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (30 μg per sample) before nitrocellulose membrane transfer (Millipore Biotech, MA, USA). The membranes were blocked with 5% skim milk and probed with primary and horseradish peroxidase (HRP)-conjugated rabbit secondary antibodies (Beyotime). To detect the signal, an enhanced chemiluminescence system (ECL, Millipore Biotech) was used. Band density was quantified using ImageJ software (http://rsb.info.nih.gov/ij/ , MD, USA) and normalized to GAPDH. The primary antibodies were as follows: EPAS1 (Abcam, ab222396, 1:2000), SLC3A2 (Abcam, ab244356, 1:1000), p-AKT (CST, #4060, 1:1000), AKT (CST, #4685, 1:1000), p Y576-FAK (Abcam, ab76120, 1:1000), FAK (Abcam, ab40794, 1:500), Cas (Abcam, ab92514, 1:1000), pY20-Cas (BioSource International, AHO0681, 1:1000), cleaved caspase-3 (CST, #9661, 1:1000), and GAPDH (CST, #5174, 1:1000).
6
Protein Extraction and Western Blot Analysis
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Protein samples derived from mouse skins and cells were extracted by RIPA lysis buffer containing phosphatases and proteases inhibitor cocktails (Roche, USA). Protein concentration was determined by BCA protein assay kit (Pierce, USA). For immunoblotting analysis, protein samples were subjected with SDS-polyacrylamide gel electrophoresis and proteins were transferred to PVDF membranes (Millipore). After being blocked, and incubated with primary antibodies and indicated HRP-coupled secondary antibody, membranes were visualized with ECL and images were captured using the Bio-Rad system. Band intensities were detected, normalized, and quantified with the Image Lab 5.0 software (Bio-Rad). The following antibodies were used in this research: NAT10 (Abcam, ab194297, 1:1000 dilution), STAT3 (CST, 30835, 1:1000 dilution), p-STAT3 (Abcam, ab76315, 1:1000 dilution), p-p65 (CST, 3033, 1:1000 dilution), p65 (CST, 8242, 1:1000 dilution), p-FAK(CST, 8556, 1:1000 dilution), FAK (Abcam, ab40794, 1:1000 dilution), Lamin B1 (Abcam, ab133741, 1:1000 dilution), poly-Ubiquitin (CST, 3936, 1:1000 dilution), Fibronectin(CST, 26836, 1:1000 dilution), α-SMA(Proteintech, 14395-1-AP, 1:1000 dilution), α-Tubulin (Beyotime, AT819, 1:5000 dilution), and GAPDH (Beyotime, AG019, 1:5000 dilution).
7
Protein Expression Analysis of Pancreatic Cancer
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Protein samples isolated from indicated pancreatic cancer cells were loaded onto SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk, incubated with primary antibodies HK2 (Anti-HK2, 1:1000, ab209847, Abcam), PKM2 (Anti-PKM2, 1:1000, ab85555, Abcam), GUT1(Anti-GUT1, 1:1000, ab115730, Abcam), PTK2 (Anti-PTK2, 1:1000, ab40794, Abcam), AKT(Anti-AKT, 1:1000, ab18785, Abcam), p-AKT(Anti-AKT1-phospho-S473, ab81283, Abcam), mTOR(1:1000, 66888-1-Ig, Proteintech), p-mTOR(Anti-mTOR-phospho S2448, ab109268, Abcam), GAPDH (1:1000, 60004-1-Ig, Proteintech), followed by reaction with HRP-conjugated secondary antibodies (1:1000, Boster). ECL Western Blotting Substrate (Beyotime) was applied for Western blot band detection. Images were visualized under Biorad Imaging Systems.
8
Protein Expression and Signaling Pathways
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Protein (40 μg) was extracted using protein lysis buffer (Beyotime, Beijing, China), separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk, followed by incubation with primary antibodies at 4 °C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h. Blots were developed using the ECL system (Amersham Biosciences, Little Chalfont, UK). The antibodies used in our study were purchased from Abcam (Cambridge, UK), including anti-β-catenin (phospho Y489) antibody (ab119801), anti-β-catenin antibody ab265591), anti-focal adhesion kinase (FAK) (phospho Y397) antibody (ab81298), anti-FAK antibody (ab40794), anti-bone morphogenetic protein (BMP2) antibody (ab214821), anti-osteocalcin (OCN) antibody (ab133612), anti-Runt-related transcription factor (RUNX2) antibody (ab264077). GAPDH was selected as internal reference gene. All experiments were repeated at least three times.
9
Western Blot Analysis of Signaling Pathways
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OS cells were treated with different concentrations of PSO for 24 h and then lysed with RIPA buffer. The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (0.45 μm). After blocking with 5% skim milk for 1 h and washing three times with Tris-buffered saline (TBS), the membranes were probed with primary antibodies at 4 °C overnight. After washing three times with TBS-Tween, the membranes were incubated with a secondary antibody (goat anti-rabbit/mouse IgG, 1:5000) at room temperature for 2 h. Protein bands were imaged and analyzed using a ChemiDoc MP Imaging System and Image Lab Software (Bio-Rad, Hercules, CA, USA). The following antibodies were used: anti-PCNA (ab92552, Abcam), anti-matrix metalloproteinase (MMP) 2 (ab92536, Abcam), anti-MMP9 (ab76003, Abcam), snail rabbit mAb (#3879, CST), bcl-2 rabbit mAb (#4223, CST), bax rabbit mAb (#41,162, CST), cleaved caspase-3 rabbit mAb (#9654, CST), β-actin rabbit mAb (#4970, CST), anti-FAK (ab40794, Abcam), anti-p-FAK (phospho Y397) (ab81298, Abcam), anti-PI3 kinase p85 alpha (ab191606, Abcam), anti-p-PI3 kinase p85 alpha (phospho Y607) (ab182651, Abcam), anti-Akt (ab179463, Abcam), and anti-p-Akt (phospho S472 + S473 + S474) (ab192623, Abcam).
10
Western Blot Analysis of Synovial Tissue
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The total protein of synovial tissue lysates and RA-FLS were extract using RIPA lysis buffer (Thermo Fisher Scientific; #8900). Protein samples were resolved on SDS-PAGE and transferred onto PVDF membranes (Millipore). Membranes were blocked in wash buffer containing 5% non-fat dry milk with gentle agitation for 1 h at room temperature. The membranes were then incubated with Antibody Dilution-diluted primary antibodies, as follows. HIF-1α antibody (1:500, GeneTex, GTX127309); RhoA antibody (1:500, Abcam, ab54835); phosphorylated (P)-RhoA antibody (1:500, Abcam, ab41435); FAK antibody (1:500, Abcam, ab40794); P-FAK antibody (1:500, Abcam, ab81298); MLC antibody (1:1000, CST, #3672); P-MLC antibody (1:1000, CST, #3675); GAPDH antibody (1:2000, Abcam, ab8245), at 4 °C overnight with gentle agitation. After washing thrice on the shaker for 10 min each time, the membranes were incubated at 1:1000 dilution of Goat anti-rabbit IgG (HRP) (1:2000, Abcam, ab6721) or anti-mouse IgG (HRP) (1:2000, Abcam, ab6728) for 2 h. Following three final 15-min washes, the enhanced chemiluminescence (ECL) detection reagent (Millipore) was placed on the membranes for 5 min before they were exposed to Hyper film ECL.
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