The protein expression in kidney tissue and two renal tubular epithelial cell lines were analyzed by western blotting. Equal amounts of protein samples were loaded on sodium dodecyl sulfate-polyacrylamide (SDS) gels for electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes and blotted with fibronectin (Cell Signaling, USA), α-SMA (abcam, UK), vimentin (Genscript, USA), E-cadherin (BD Biosciences, Canada), p-Smad2/3 (Cell Signaling, USA), Smad2/3 (Cell Signaling, USA), PAI-1 (Cell Signaling, USA), Collagen I (Santa Cruz, USA), β-actin (Santa Cruz, USA) and GAPDH (Santa Cruz, USA) primary antibodies, followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. The proteins were detected using western lightning series HRP chemiluminescent substrates and captured on light-sensitive X-ray film.
Vimentin
Manufactured by GenScript
Sourced in United Kingdom, United States
Vimentin is a type III intermediate filament (IF) protein that is commonly used as a marker for mesenchymal cells and is involved in maintaining the structural integrity of cells. It is an essential component of the cytoskeleton and plays a role in various cellular processes, such as cell migration, adhesion, and signaling.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (5 mentions)
E cadherin, by GenScript (3 mentions)
Fibronectin, by Cell Signaling Technology (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
α sma, by Abcam (1 mentions)
Collagen 1, by Santa Cruz Biotechnology (1 mentions)
Pai 1, by Cell Signaling Technology (1 mentions)
P smad2 3, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by BD (1 mentions)
Enhanced chemiluminescence blotting analysis system, by Cytiva (1 mentions)
Total histone 3, by Cell Signaling Technology (1 mentions)
Nuclear extraction kit, by Merck Group (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Twist, by Abcam (1 mentions)
H3k27me3, by Cell Signaling Technology (1 mentions)
Lamin b1, by Santa Cruz Biotechnology (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Snail, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse immunoglobulin g antibody, by Santa Cruz Biotechnology (1 mentions)
5 protocols using vimentin
1
Protein Expression Analysis in Kidney Cells
2
Western Blot Analysis of Protein Expression
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Whole-cell or nuclear protein extracts were prepared using the M-Per Mammalian Protein Extraction Reagent (Pierce Biotechnology, Woburn, MA) or the Nuclear Extraction Kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions. Total protein (30 μg) and nuclear protein (10 μg) were loaded onto 10–20% SDS-PAGE gels, electrophoresed, and then transferred to nitrocellulose membranes. Antigen-antibody complexes were detected using the enhanced chemiluminescence blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK). The following antibodies were used: EZH2 (Cell Signaling; 5246), H3K27me3 (Cell Signaling; 9733), total histone 3 (Cell Signaling; 9715), E-cadherin (GenScript; A01589), Vimentin (GenScript; A01189), Twist (Abcam; ab50887), p-AKT (Santa Cruz; sc-293125), AKT (Santa Cruz; sc-1618), GAPDH (Santa Cruz; sc-47724), YY1 (Santa Cruz; sc-7341) and lamin B1 (Santa Cruz; sc-20682). GAPDH (whole cell lysate) and lamin B (nuclear protein) were applied as loading controls. Primary antibodies were used at a dilution of 1:1000.
3
Immunoblot Analysis of EMT Markers
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Cells were harvested 24 h after transfections. Equal amounts of protein lysates (30 μg) were separated by 10 % SDS-PAGE for immunoblots with antibodies to Snail (Abcam), E-cadherin (GenScript), N-cadherin (BD Biosciences), Vimentin (GenScript) and GAPDH (Santa Cruz). Primary antibodies were used at a dilution of 1:1000. A horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin-G antibody was used as the secondary antibody (1:5000; Santa Cruz). Signals were detected using enhanced chemiluminescence reagents (Amersham Biosciences).
4
Protein Expression Analysis by Western Blot
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A total of 30 µg protein was separated by 10% SDS-PAGE, and membranes were incubated with primary antibodies against human MMSET (HPA015801, Sigma-Aldrich, St. Louis, MO), Twist1 (ab50887, Abcam, Cambridge, MA), Vimentin (A01189, GenScript, Edison, NJ), E-cadherin (A01589, GenScript, Edison, NJ), H3K9me2 (4658, Cell Signaling, Danvers, MA), total histone H3 (4499, Cell Signaling, Danvers, MA) and GAPDH (sc-47778, Santa Cruz, Santa Cruz, CA), followed by HRP-conjugated secondary antibody and developed using the ECL reagent (Amersham, Poole, UK).
5
Western Blotting of Cell Proteins
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Protein extracts for western blotting were prepared in M-Per Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, United States), separated by SDS-polyacrylamide gels, and then transferred to PVDF membrane (GE Healthcare Life Sciences, Piscataway, NJ, United States). Membranes were incubated with primary antibodies including: PD-L1 (1:1000, clone E1L3N, Cell Signaling), MCL-1 (1:1000, #4572, Cell Signaling), ZO-1 (1:1000, #5406, Cell Signaling), Vimentin (1:1000, A01189, GenScript, Edison, NJ, United States), and GAPDH (1:3000, sc-47724, Santa Cruz Biotechnologies, Santa Cruz, CA, United States), and then with HRP-conjugated secondary antibody. Finally, blots were developed with the ECL detection kit (Amersham Pharmacia Biotech, United Kingdom). GAPDH served as the loading control. Immunoblot images were quantified using the NIH Image software.
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