Total protein was isolated from primary cultured chondrocytes using RIPA lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris pH 8.0, 0.2% sodium dodecyl sulfate, and 5 mM NaF) containing a protease and phosphatase inhibitor cocktail (Roche, Madison, WI, USA). MMP3 and MMP13 were isolated from chondrocyte culture-conditioned medium using trichloroacetic acid, as previously described [33 (link)]. The protein extracts were then separated by SDS-PAGE and transferred onto a PVDF membrane by western blotting. The following antibodies were used for western blotting: anti-MMP3 (ab52915; Abcam, Cambridge, UK); mouse anti-MMP13 (ab51072; Abcam); mouse anti-Erk1/2 (610408; Becton Dickinson, Bergen County, NJ, USA); mouse anti-IκB (9242; Cell Signaling Technology (CST),Danvers MA, US); mouse anti-p65 (#6956; CST); mouse anti-pp65 (#13346; CST); mouse anti-p38 (#9212; CST); mouse anti-pp38 (#9215S; CST); mouse anti-c-Jun N-terminal kinase (JNK) (#9252S; CST); mouse anti-pJNK (#9251S; CST), and mouse anti-pErk (#9101S; CST). The protein bands were subsequently detected on the blotted membranes using a secondary antibody and the SuperSignal West Dura kit (Thermo Scientific, Waltham, MA, USA). Erk1/2 was used as a house-keeping protein during the subsequent analysis.
Supersignal west dura kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperSignal West Dura kit is a chemiluminescent detection system for Western blotting. It is designed to provide sensitive and reliable detection of proteins on membranes.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Bio-Rad (7 mentions)
Dc protein assay, by Bio-Rad (6 mentions)
Mouse anti erk1 2, by BD (5 mentions)
Mouse anti iκb, by Cell Signaling Technology (4 mentions)
Chemidoc mp visualization system, by Bio-Rad (4 mentions)
Image lab, by Bio-Rad (4 mentions)
Goat anti cox 2 sc 1745, by Santa Cruz Biotechnology (4 mentions)
Bca assay, by Thermo Fisher Scientific (4 mentions)
Ab51072, by Abcam (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Alphaeasefc 4, by Bio-Techne (3 mentions)
β actin, by Merck Group (3 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Chemidoc mp system, by Bio-Rad (3 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Ab52915, by Abcam (2 mentions)
60 protocols using supersignal west dura kit
1
Chondrocyte Protein Isolation and Analysis
2
Immunoblotting of Pancreatic ESC Proteins
3
Collagen IV and Fibronectin Signaling
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Cells were plated for 60 minutes on collagen IV- or fibronectin-coated flasks. Cell lysates were then collected and immunoblots were performed using the Phospho- motif primary antibodies provided in the KinomeView Kit (Cell Signaling Technology #9812). Appropriate secondary antibodies were used to detect the signaling motifs on each immunoblot. Final visualization was achieved via incubation with Supersignal West Dura kit (Thermo Scientific) and images were taken with Fuji digital film imaging station.
4
Western Blot Analysis of Brain Extracts
5
Western Blot Analysis of Mitochondrial Proteins
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Mitochondrial fractions were obtained using the Mitochondria Isolation Kit for Tissue (89801, ThermoFisher Scientific, Waltham, MA). Protein concentrations were measured with the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were loaded into lanes of Criterion TGX (Tris–Glycine eXtended) Stain-Free PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA. The gels were electrophoresed and activated using a ChemiDoc MP Visualization System (Bio-Rad Laboratories, Hercules, CA, USA). The protein was then transferred to a PVDF membrane. The membranes were then imaged using a ChemiDoc MP Visualization System to obtain an assessment of proper transfer and to obtain total protein loads. The membranes were then blocked and probed with primary antibodies overnight at 4 °C. Immunoblots were next processed with secondary antibodies (Cell Signaling) for 1 h at room temperature. Immunoblots were then probed with a Super Signal West Dura kit (Thermo Fisher Scientific) to visualize signal, followed by visualization using a ChemiDoc MP Visualization System (Bio-Rad Laboratories, Hercules, CA, USA). Data were analyzed using Image Lab (Bio-Rad Laboratories, Hercules, CA, USA)58 (link). Uncropped images of immunoblots are depicted in Supplemental Figs. 1 –5 .
6
Western Blot and Reverse Phase Protein Array
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For western blotting, cells were collected on ice using a scraper, washed in PBS and lyzed in RIPA buffer (50 mM Tris-HCl, pH7.4; 1% NP-40; 0.5% sodium-deoxycholate; 0.1% SDS; 150 mM NaCl) supplemented with phosphatase and protease inhibitors (Roche Applied Science, Mannheim, Germany) and 50 µM OGA inhibitor (PUGNAc, Sigma Aldrich). Following ultra-sonification, protein concentration of the samples was measured using the BCA protein assay kit (Pierce™, Thermo Scientific). 15–20 µg of protein was loaded per each gel lane. Membranes were visualized using the super signal West Dura kit (Thermo Scientific) in the Syngene G:Box instrument (Syngene, Cambridge, UK) with GeneSnap software. Densitometry was performed using ImageJ.
Reverse phase protein array (RPPA) was performed at the core facility of functional proteomics in MD Anderson Cancer Center, University of Texas, Houston TX. Protein lysate samples were prepared for analysis in accordance with the core facility’s instructions (available athttps://www.mdanderson.org/research/research-resources/core-facilities/functional-proteomics-rppa-core.html ). Protein concentration was adjusted to 1.5 µg/ml, and 35–50 µl of each sample was submitted for analysis.
Reverse phase protein array (RPPA) was performed at the core facility of functional proteomics in MD Anderson Cancer Center, University of Texas, Houston TX. Protein lysate samples were prepared for analysis in accordance with the core facility’s instructions (available at
7
Quantitative Western Blot Analysis
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Cytosolic protein extracts were isolated from liver tissue and separated in SDS-PAGE as detailed before [34 (link)]. After transferring proteins to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA), and incubating them with primary antibodies detecting cleaved caspase 3 (Cell Signaling Technology, Massachusetts, USA) or β-actin (Santa Cruz Biotechnology, Dallas, Texas, United States), bands were detected using Super Signal West Dura kit (Thermo Fisher Scientific, Waltham, MA, USA) [34 (link), 36 (link)] and analyzed using the ChemiDoc XRS System with Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).
8
Chondrocyte Protein Extraction and Analysis
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Whole protein was extracted from the primary cultured chondrocytes using RIPA lysis buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris/HCl (pH 8.0), 0.2% SDS, and 5 mM NaF with addition of protease and phosphatase inhibitor mixture (Roche, Madison, WI, USA). Total proteins were separated by SDS-PAGE, and Western blotting analysis was performed. The following antibodies were used: Goat anti-COX-2 (sc-1745; Santa Cruz, Dallas, TX, USA); mouse anti-ERK1/2 (610408; Becton Dickinson, NJ, USA); mouse anti-IκB (9242; Cell Signaling Technology, Danvers, MA, USA); mouse anti-p65 (8242; CST; mouse anti-phospho-p65 (3033; CST mouse anti-p38 (#9212; CST), mouse anti-pp38 (#9215S; CST); mouse anti-c-Jun N-terminal kinase (JNK) (#9252S; CST); mouse anti-pJNK (#9251S; CST); mouse anti-pErk (#9101S; CST). Each signal was visualized using the SuperSignal West Dura Kit (Thermo Scientific, Waltham, MA, USA). Density analysis (AlphaEase FC 4.0; Alpha Innotech, San Leandro, CA, USA) was used to quantify the relevant band intensities. Extracellular signal-regulated kinase (ERK) was used as the loading control.
9
Protein Extraction and Western Blot Analysis
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Cells were lysed in buffer containing 40 mM HEPES, 120 mM sodium chloride, 10 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 1 mM EDTA, 50 mM sodium fluoride, 0.5 mM sodium orthovanadate and 1% Triton X-100. Cell lysates were gently resuspended and incubated at 4°C with gentle rocking for 40 min to 1 h, followed by microcentrifugation for 10 min at 4°C. The supernatants were transferred to new tubes and protein concentrations were determined. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes, and immunoblotting was performed using the antibodies described above. Detection by enhanced chemiluminescence was with a SuperSignal West Dura kit (Thermo Fisher Scientific, Rockford, IL) and a Bio-Rad Fluor-S Max Multi-Imager.
10
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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