After culturing in osteogenic inducing medium containing different concentrations of puerarin (0 and 10−5 mol/L) for 14 days, BMSCs were lysed with RIPA lysis buffer containing 1% PMSF (Solarbio, Beijing, China). The total collected protein concentrations were quantified by a BCA protein assay kit. All protein samples (20μg) were denatured and separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto 0.45-μm polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA). Afterwards, the membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated with primary antibodies that recognized β-catenin (1:800; Cell Signaling Technology, Danvers, MA, USA), GAPDH, ALP, Runx2, Collagen I (Col I) (1:1000; Abcam, Cambridge, MA, USA) overnight at 4 ℃. After washing in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with a secondary antibody (Absin, Shanghai, China) solution at 37 ℃ for 1 h. Secondary antibodies were selected based on the source of primary antibodies. An enhanced chemiluminescent substrate kit (Millipore) and a chemical imaging system (Amersham Imager 600; GE Healthcare, Little Chalfont, UK) were used to detect immunoreactive proteins. GAPDH was used as internal reference.
Collagen 1 col 1
Manufactured by Abcam
Sourced in United States, United Kingdom
Collagen I (Col I) is a structural protein found in the extracellular matrix of various tissues, including skin, bone, and tendon. It is the most abundant type of collagen in the human body. Col I plays a crucial role in providing tensile strength and supporting the integrity of these tissues.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Abcam (2 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Runx2, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Enhanced chemiluminescent substrate kit, by Merck Group (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Biotinylated secondary antibody, by Abcam (1 mentions)
Masson s trichrome staining, by Wuhan Servicebio Technology (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Vimentin, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
Fibronectin fn, by Abcam (1 mentions)
Collagen 4 col 4, by Abcam (1 mentions)
α smooth muscle actin α sma, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Chemidoc mp chemiluminescence gel imaging system, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
5 protocols using collagen 1 col 1
1
Evaluating Osteogenic Potential of Puerarin
2
Quantitative Analysis of Kidney Fibrosis
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The kidney tissues were fixed in 4% v/v paraformaldehyde for 48 h, dehydrated using a graded ethanol series, embedded in paraffin, and sectioned (5 μm). For immunohistochemical staining, antigen retrieval was performed using a citrate solution after deparaffinization and rehydration. The sections were blocked with 5% v/v goat serum and then incubated with the following primary antibodies at 4 °C overnight: collagen-I (col-I; Abcam, Cambridge, England) and α-smooth muscle actin (α-SMA; Abcam). The sections were then incubated with biotinylated secondary antibodies (Abcam) for 2 h at RT and streptavidin-peroxidase complex for 1 h at RT then visualized with a 3,3′-diaminobenzidine system according to the manufacturer's protocol. The sections were counterstained with hematoxylin and dehydrated. The dehydrated sections were mounted using neutral resin. Masson's trichrome staining was performed according to the manufacturer's instructions (Servicebio). Quantification of collagen-I-, α-SMA-, and Masson-positive areas was performed by taking random cortical images (original magnification power × 40, six fields per kidney) of renal cortex from each mouse and counting the percentages of the positivelystained areas in each microscopic field.
3
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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After extracting the proteins in cells using RIPA buffer (Invitrogen), the protein concentration was detected. After separating the protein samples by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to polyvinylidene fluoride membranes (Millipore, USA). Subsequently, after soaking for 2 h in 5% skim milk, the membranes were incubated with primary antibodies overnight at 4°C. Then, the membranes were coated with rabbit secondary antibody (1:4,000; Abcam, UK) for 2 h at 37°C. The protein bands were observed using enhanced chemiluminescence reagents (Millipore). The primary antibodies included E-cadherin (1:1,000; Abcam), vimentin (1:1,000; Abcam), α-smooth muscle actin (α-SMA) (1:1,000; Abcam), fibronectin (FN) (1:1,000; Abcam), collagen I (Col I) (1:1,000; Abcam), collagen IV (Col IV) (1:1,000; Abcam), BMP7 (1:1,000; Abcam), and GAPDH (1:1,000; Abcam).
4
Protein Isolation and Western Blot Analysis of Bone Tissues
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Bone tissues
were placed in a mortar, and liquid nitrogen was added, allowing the
callus to be ground to fine particles with a pestle. Subsequently,
the tissue was transferred to a 1.5 mL EP tube for protein isolation.
Total protein was extracted from cells and tissues by RIPA (Beyotime,
P0013B, Shanghai, China) with 1% protease inhibitor cocktail for general
use, 100× (Beyotime, P1005, Shanghai, China). Protein (20 μg)
was separated on a denatured sodium dodecyl sulfate-polyacrylamide
gel and transferred to a polyvinylidene fluoride (PVDF) membrane.
The PVDF membrane was blocked with defat mike (5% dissolved in TBS-T)
for 2 h and then incubated with rabbit polyclonal ALP (1:1000, Abcam),
Runx2 (1:1000, Abclonal), OCN (1:500, Abcam), collagen I (Col1) (1:3000,
Abcam), GAPDH (1:1000, Abclonal), SSRP1 (1; 1000, Proteintech), and
β-catenin (1:1000, Abclonal) for 16 h at 4 °C. After being
washed three times, the membranes were followed by incubation with
horseradish peroxidase-coupled goat antirabbit IgG H&L for 1 h
at 37 °C. The blotting membrane was treated with an ECL chemiluminescent
substrate (Biosharp, BL520A, Shanghai, China) and visualized using
a ChemiDoc MP chemiluminescence gel imaging system (Bio-Rad,1708280,
California, USA).
were placed in a mortar, and liquid nitrogen was added, allowing the
callus to be ground to fine particles with a pestle. Subsequently,
the tissue was transferred to a 1.5 mL EP tube for protein isolation.
Total protein was extracted from cells and tissues by RIPA (Beyotime,
P0013B, Shanghai, China) with 1% protease inhibitor cocktail for general
use, 100× (Beyotime, P1005, Shanghai, China). Protein (20 μg)
was separated on a denatured sodium dodecyl sulfate-polyacrylamide
gel and transferred to a polyvinylidene fluoride (PVDF) membrane.
The PVDF membrane was blocked with defat mike (5% dissolved in TBS-T)
for 2 h and then incubated with rabbit polyclonal ALP (1:1000, Abcam),
Runx2 (1:1000, Abclonal), OCN (1:500, Abcam), collagen I (Col1) (1:3000,
Abcam), GAPDH (1:1000, Abclonal), SSRP1 (1; 1000, Proteintech), and
β-catenin (1:1000, Abclonal) for 16 h at 4 °C. After being
washed three times, the membranes were followed by incubation with
horseradish peroxidase-coupled goat antirabbit IgG H&L for 1 h
at 37 °C. The blotting membrane was treated with an ECL chemiluminescent
substrate (Biosharp, BL520A, Shanghai, China) and visualized using
a ChemiDoc MP chemiluminescence gel imaging system (Bio-Rad,1708280,
California, USA).
5
Western Blot Analysis of Fibrosis Markers
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Thirty micrograms of protein were dissolved in SDS-PAGE gel, then transferred onto polyvinylidene difluoride membranes (Sigma-Aldrich) by electroblotting. After electroblotting, the membranes were blocked for 1 hour at room temperature with 5% blocking solution, the required bands were cut according to marker, and THBS1 antibodies (1:1000, Cell Signaling Technology, Cat# 37879), TGF-β1 (1:1000, Abcam, ab92486), α-SMA (1:500, Abcam, ab5694), Collagen I (Col-1, 1:1000, Abcam, ab6308), Cleavage Caspase 3 (1:1000, Cell Signaling Technology, Cat# 9661), and β-Actin (1:1000, Abcam, ab8227) were incubated overnight at 4°C. The second antibody (1:5000) was incubated at room temperature for 1 hour. Immunoreactivity was measured using the Western Lighting Ultra Kit (ECL, Pierce Technology, Shanghai, China).
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