nm‐cFib were analysed in cold buffer (20 mM HEPES, 150 mM NaCl, 10% [v/v] glycerol, 0.5% [v/v] NP‐40, 1 mM EDTA, 2.5 mM DTT, 10 μg/L aprotinin, leupeptin, pepstatin A, 1 mM PMSF and Na3VO4) and total protein content determined with the Bicinchonic Protein assay kit (Merck Group, Vimodrone, Italy); 20 μg protein were resolved on SDS‐polyacrylamide gels and electro‐transferred to a PVDF membrane (Merck Group). To quantify shed TLR4 (sTLR4), 100 μL of conditioned media were concentrated to a final volume of 25 μL and run on SDS‐polyacrilamide gels. Blots were hybridized with primary antibodies against Postn (rabbit polyclonal 19899‐1‐AP, Proteintech, UK), TLR4 (mouse monoclonal sc‐293072, Santa Cruz Biotechnology) and NRF2 (rabbit polyclonal, C‐20, Santa Cruz Biotechnology) and incubated with corresponding horseradish peroxidase secondary antibodies (Cell Signaling Technology). Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Merck). Band intensities were determined using Alliance system (Uvitec, Cambridge, UK).
Alliance system
Manufactured by Uvitec
Sourced in United Kingdom
The Alliance system is a lab equipment product that serves as a core function. It provides a platform for essential laboratory operations without further interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (4 mentions)
Bicinchonic protein assay kit, by Merck Group (3 mentions)
Sc 293072, by Santa Cruz Biotechnology (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
P nfkbp65 ser536, by Santa Cruz Biotechnology (1 mentions)
Anti p smad2, by Cell Signaling Technology (1 mentions)
Anti p smad3, by Cell Signaling Technology (1 mentions)
Anti phospho smad2, by Cell Signaling Technology (1 mentions)
Anti phospho smad3, by Cell Signaling Technology (1 mentions)
5 protocols using alliance system
1
Quantification of Shed TLR4 and Related Proteins
2
Immunoblotting Analysis of Cell Signaling Pathways
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Cells were lysed in cold buffer (20 mM HEPES, 150 mM NaCl, 10% [v/v] glycerol, 0.5% [v/v] NP-40, 1 mM EDTA, 2.5 mM DTT, 10 µg/L aprotinin, leupeptin, pepstatin A, 1 mM PMSF, and Na3VO4). Protein concentration was determined by using the Bicinchonic Protein assay kit (Merck Group, Vimodrone, Italy) and 10–20 μg were resolved on SDS-polyacrylamide gels and electro-transferred to a PVDF membrane (Merck Group). Blots were probed using anti CCL2/MCP-1 polyclonal antibody (Novus Biologicals, Space Import-Export s.r.l., Milan, Italy), anti phospho-ERK1(T202/Y204)/ERK2 (T185/Y187) polyclonal antibody (R&D Systems, Space Import-Export s.r.l., Milan, Italy), anti phospho-p38 MAP Kinase (T180/Y182) polyclonal antibody (R&D Systems), anti phospho-JNK (Thr 183/Tyr 185) (Santa Cruz Biotechnology), reprobed with β-actin or ERK, p38, JNK (Santa Cruz Biotechnology) and incubated in horseradish peroxidase secondary antibodies (Cell Signaling Technology). Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Merckgroup). Band intensities were determined using Alliance system (Uvitec, Cambridge, UK).
3
Protein Extraction and Immunoblotting
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Cell layers were lysed in cold buffer (20 mM HEPES, 150 mM NaCl, 10% [v/v] glycerol, 0.5% [v/v] NP-40, 1 mM EDTA, 2.5 mM DTT, 10 ug/L aprotinin, leupeptin, pepstatin A, 1 mM PMSF, and Na3VO4). Protein concentration was determined by using the Bicinchonic Protein assay kit (Merck, Milan, Italy) and 10–20 µg were resolved on SDS-polyacrylamide gels and electro-transferred to a PVDF membrane (Merck). Blots were probed using anti Myostatin polyclonal antibody (Proteintech Europe), anti-phospho-ERK1(T202/Y204)/ERK2 (T185/Y187) (R&D Systems, Space Import-Export s.r.l., Milan, Italy), p-NFkB p65 (Ser 536) (Santa Cruz Biotechnology), anti p-SMAD2 (Cell Signaling Technology, Euroclone, Milan, Italy), anti p-SMAD 3 (Cell Signaling Technology), p16ink4a (St John’s Laboratory, D.B.A. Italia s.r.l.) and reprobed with β-actin or ERK (Santa Cruz Biotechnology) or SMAD 2,3 (Cell Signaling Technology) and incubated in horseradish peroxidase secondary antibodies (Cell Signaling Technology).Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Merck). Band intensities were determined using an Alliance system (Uvitec, Cambridge, UK).
4
Immunoblot Analysis of SMAD Signaling
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A7r5 cell layers were lysed in cold buffer (20 mM HEPES, 150 mM NaCl, 10% [v/v] glycerol, 0.5% [v/v] NP-40, 1 mM EDTA, 2.5 mM DTT, 10 ug/L aprotinin, leupeptin, pepstatin A, 1 mM PMSF, and Na3VO4). Protein concentration was determined by using the Bicinchoninic Protein Assay Kit (Merck Group, Vimodrone, Italy), and 10–20 µg of protein concentration were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to a PVDF membrane (Merck Group, Vimodrone, Italy). Blots were probed using anti-phospho-SMAD 2, anti-phospho-SMAD 3, and SMAD 2/SMAD 3 polyclonal antibody (Cell Signaling Technology, EuroClone, Pero, Italy) and incubated in horseradish peroxidase secondary antibodies (Cell Signaling Technology, EuroClone, Pero, Italy). Immunoblots were developed with Immobilon Western Chemiluminescent HRP Substrate (Merck Group, Vimodrone, Italy). Band intensities were determined using Alliance system (Uvitec, Cambridge, UK).
5
Western Blot Analysis of Frozen Renal Tissue
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Frozen renal tissue was lysed in RIPA buffer with cOmplete™ Protease Inhibitor Mixture. Briefly, equal amounts of proteins were mixed with reducing sample buffer and denatured for 10 minutes at 100°C. Protein samples were then resolved by 10% SDS PAGE and electro-transferred to nitrocellulose membranes. Nonspecific binding was blocked for 1 h at room temperature with 3% BSA in TBST buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20). Membranes were exposed to primary antibodies overnight at 4°C and incubated with a secondary peroxidase-conjugated antibody for 1 h at room temperature (Table 2 ). Detection was performed using a chemiluminescence substrate with Alliance system (UVItec).
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