Total protein extraction from human dermal fibroblasts or A549 cells was performed using an extraction kit (101 Bio, Mountain View, CA, USA). Protein concentration was determined using a spectrophotometer and bicinchoninic acid protein assay kit (TaKaRa Bio, Shiga, Japan). Aliquots of protein from each sample were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a Mini-PROTEAN TGX Precast gel and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was incubated with the appropriate primary antibody and fluorescently labeled secondary antibodies. The following antibodies were used: anti-COL1A2 (1:1000, Abcam), anti-FN1 (1:1000, Abcam), anti-αSMA (1:1000, Abcam), anti-p-Smad3 (1:1000, Invitrogen), anti-calpain 1 (1:1000, Abcam), anti-calpain 2 (1:1000, Abcam), anti-E-cadherin (1:1000, Abcam), anti-GAPDH hFAB rhodamine (Bio-Rad Laboratories), and anti-tubulin hFAB rhodamine (Bio-Rad Laboratories). In addition, anti-mouse or anti-rabbit fluorescent-labeled secondary antibodies (all Bio-Rad Laboratories) were used as secondary antibodies. Blots were scanned and quantified using a ChemiDoc imaging system (Bio-Rad Laboratories). The quantified band intensities were normalized to those of GAPDH or tubulin.
Anti fn1
Manufactured by Abcam
Sourced in United States
Anti-FN1 is a primary antibody that binds to the FN1 (Fibronectin 1) protein. FN1 is an extracellular matrix glycoprotein involved in cell adhesion and migration processes. The Anti-FN1 antibody can be used in various immunoassay techniques to detect and study the FN1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti α sma, by Abcam (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Anti psmad3, by Thermo Fisher Scientific (1 mentions)
Hfab rhodamine anti tubulin, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Takara Bio (1 mentions)
Anti col1a2, by Abcam (1 mentions)
Anti calpain 1, by Abcam (1 mentions)
Anti fsp 1, by Merck Group (1 mentions)
Criterion tgx gel, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Anti smad2 3, by Cell Signaling Technology (1 mentions)
Anti opn, by R&D Systems (1 mentions)
Anti phospho smad2 ser465 467 smad3 ser423 425, by Cell Signaling Technology (1 mentions)
Anti tgf β1, by Santa Cruz Biotechnology (1 mentions)
Anti col1, by Abcam (1 mentions)
6 protocols using anti fn1
1
Quantitative Protein Analysis of Dermal Fibroblasts
2
Antibody Sourcing for Protein Analysis
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Mouse monoclonal anti-PrP (ICSM18) was obtained from D-Gen Limited (London, UK). Rabbit polyclonal anti-Fn1 (Cat# ab2413) was obtained from Abcam. Rabbit polyclonal anti-Lrrn4/Lrch4 (Cat# GTX1 12459) was purchased from GeneTex. Rabbit polyclonal antibody anti-IL11ra1 (Cat# 10264-1-AP) was purchased from Proteintech. Rabbit polyclonal anti-Itga8 (Cat# sc-25713) and rat monoclonal anti-NCAM (clone H28-123; Cat# sc-59934) were purchased from Santa Cruz. Rabbit polyclonal anti-CHGA (Cat# HPA017369) was obtained from Sigma. Mouse monoclonal anti-HS antibodies JM403 (Cat# 370730-1) and 10E4 (Cat# 370255-1) were obtained from Seikagaku (AMS Biotechnology, Abingdon OX14 4SE, UK). Mouse monoclonal anti-IQGAP2 (clone A2) was kindly provided by Prof. George S. Bloom. This antibody, a murine monoclonal IgG2a antibody against IQGAP2, was produced by hybridoma cells derived by fusion of Sp2/0-Ag14 myeloma cells with splenic lymphocytes isolated from a male A/J mouse that was immunised with purified, recombinant his-tagged human IQGAP2. The IQGAP2 was expressed in baculovirus-infected high five insect cells and purified by nickel affinity chromatography. The IQGAP2 coding sequence in the baculovirus was obtained by PCR amplification from a human brain cDNA library.
3
Western Blot Analysis of Lung Tissue
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Randomly selected lung tissue samples were homogenized and lysed in T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific), whereas cultured primary mouse lung fibroblasts were lysed in RIPA Lysis Buffer (Santa Cruz). Whole protein extract (10, 20, or 30 μg) was resolved on a 4–15% or 8–16% Criterion TGX Gel (Bio-Rad, Hercules, CA, USA). Actin was examined as a loading control. Representative blotting images were presented. The primary antibodies used for immunoblotting were anti-OPN (R&D Systems), anti-FN1 (Abcam), anti-FSP1 (EMD Millipore), anti-TGF-β1 (Santa Cruz), anti-Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (Cell Signaling Technology), anti-Smad2/3 (Cell Signaling Technology), and anti-Actin (Santa Cruz) antibodies.
4
Western Blot Analysis of Senescence and ECM Proteins
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The protein concentration in the supernatant was determined by BCA assay. Aliquots of 80 μg protein were separated in SDS-PAGE and transferred onto nitrocellulose membranes by an electric transfer (BIORAD Inc., USA). The membranes were blocked with 5% milk PBS-Tween20 (PBST) and incubated with primary antibody including: anti-Sesn1 (Abcam, USA), anti-Sesn2 (Santa Cruz Biotechnology, USA), anti-Sesn3 (Abcam, USA), anti-COL I (Abcam, USA), anti-COL III (Abcam, USA), anti-FN1 (Abcam, USA), and anti-Actin (Cell Signaling, USA) antibodies. They were then incubated with Alexa Fluor 700 antibodies (Molecular Probes, USA). Images were captured on an Odyssey Infrared Imaging System (LI-COR Biosciences, USA), and quantified using Odyssey v1.2.
5
Protein Expression Analysis of Stem Cells
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Total protein was derived from the rBMSCs and hGFs on the JEM membrane using RIPA buffer (Beyotime Biotechnology Inc.) on the indicated days, and the total protein concentration was evaluated using a BCA protein assay kit (Bestbio, China). Proteins were separated using electrophoresis on a polyacrylamide gel plate and subsequently transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA and incubated with anti-RUNX2, anti-OPN, anti-OCN, anti-COL I, anti-LC3, anti-PINK1, anti-Parkin, anti-Fak, anti-Fn1, anti-Complex I, Complex II, Complex III, Complex IV, Complex V (Abcam, Inc., Cambridge, UK) overnight at 4 °C. The membranes were then washed with TBST solution thrice, and incubated with horseradish peroxidase-conjugated secondary antibodies before visualization. Protein expression levels were normalized to that of GAPDH. The protein bands were visualized using enhanced chemiluminescence (ECL) reagent (Beyotime Biotechnology Inc.). Protein levels were quantified using Image J analysis software.
6
Western Blot Analysis of Fibrosis Markers
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RIPA (Beyotime, China) was used for harvesting cell lysates, and BCA protein assay kit (Beyotime, USA) was used for determining the concentration of protein samples. Same amounts of proteins were separated by SDS-PAGE gels (10%) and transferred to PVDF membranes. After 2 h of blocking in 5% Bovine Serum Albumin (BSA, Thermofisher, USA), PVDF membranes were incubated in primary antibodies under the temperature of room (24°C) for 2 h. After that, PVDF membranes were washed 3 times in 15 min with TBST and incubated in horseradish peroxidase-conjugated secondary antibody (Beyotime, China) for 1 hunder 24°C. Finally, an enhanced chemiluminescence (ECL, Vazyme, Nanjing, China) was used for imaging western blots. Image data were analyzed by ImageJ software (NIH, MD, USA). The antibodies used in this experiment are listed as below Anti-FN-1 (Rabbit, 1:3000, Abcam, MA, USA), anti-Col-1a1 (Rabbit, 1:1000, Abclonal, Wuhan, China), anti-α-SMA (Rabbit, 1:1000, Abcam, MA, USA), anti-E2F1 (Rabbit, 1:1000, Abclonal, Wuhan, China), anti-CCNE2 (Rabbit, 1:1000, Abclonal, Wuhan, China), anti-GAPDH (Rabbit, 1:3000, CST, MA, USA) for western-blot.
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