The relevant protein levels of TGF-β1, Smad3, P-Smad3, Smad7, α-SMA and collagen-1 in vitro were measured by western blot. Cells incubated and treated in 6-well plates were lysed by 200 μL RIPA lysis buffer containing protease inhibitor (PMSF, 1 mM) and phosphatase inhibitor (TIANDZ 80809-1, 1%) on ice-bath for 30 min. And the lysate was centrifuged at 10000 rpm for 10 min at 4 °C, the supernatant was collected and the proteins were quantified by BCA protein assay kit (Beyotime Biotechnology, China). Membranes were incubated with primary antibodies (anti-TGF-β1, 1:1000, Abcam, ab64715; anti-Smad3, 1:1000, Abcam, ab40854; anti-P-Smad3, 1:400, Bioss, BS-3425R; anti-Smad7, 1:400, Boster, BA1399; anti-α-SMA, 1:400, Boster, BM0002; anti-collagen1, 1:400, Boster, BA0325) overnight at 4 °C and mouse monoclonal anti-β-actin (1:500, Sinipept, 41302M) was used for loading control. After that HRP-goat-anti-mouse antibody (1:1000, World Biotech, WH-004) was incubated with membranes of TGF-β1, α-SMA and β-actin for 1 h at room temperature; HRP-goat-anti-rabbit antibody (1:1000, CW-biotech, cw-0103) with Smad3, P-Smad3, Smad7 and collagen-1 respectively. Immunoblot were visualized with Bio-Rad and recorded by Bio-Rad detection system, and gray density analysis was completed with the Image J program.
Ba0325
Manufactured by Boster Bio
Sourced in China, United States
The BA0325 is a laboratory centrifuge designed for general-purpose applications. It is capable of processing multiple samples simultaneously at adjustable speeds and times.
Automatically generated - may contain errors
Lab products found in correlation
Jf0582, by Huabio (3 mentions)
Bm0002, by Boster Bio (2 mentions)
Ab955, by Abcam (2 mentions)
Ba1771, by Boster Bio (2 mentions)
Ba0752, by Boster Bio (2 mentions)
Ba0290, by Boster Bio (2 mentions)
Bs 10196r, by Bioss Antibodies (2 mentions)
Upright fluorescence microscope, by Leica (2 mentions)
Goat anti rabbit igg, by Abbkine (2 mentions)
Ba0326, by Boster Bio (2 mentions)
Ab5694, by Abcam (2 mentions)
Ab64715, by Abcam (1 mentions)
Ab40854, by Bioss Antibodies (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab128507, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab152099, by Abcam (1 mentions)
Ab32515, by Abcam (1 mentions)
Anti atg7, by Cell Signaling Technology (1 mentions)
16 protocols using ba0325
1
In vitro Protein Expression Analysis
2
Liver Tissue Analysis with Imaging
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Fresh liver tissue was fixed with 4% paraformaldehyde and paraffin embedded, and then the sections (4 μm) of liver were stained with hematoxylin and eosin, Sirius Red, and Masson. The red area in Sirius staining, considered the fibrotic area, was assessed by computer-assisted image analysis with ImageJ software V1.8.0.112 (National Institutes of Health, Bethesda, MD). Immunofluorescence was used for checking the expression of CD68 (ab955; Abcam, Cambridge, MA; diluted 1:500), Collagen I (BA0325; Boster Biological Technology, Wuhan, China; diluted 1:100) and TGF-β1 (bsm33345; Bioss, Beijing, China; diluted 1:100). DAPI (Molecular Probes, Eugene, OR) was used to label cell nuclei.
3
Immunohistochemical Analysis of PDR Membranes
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Eight PDR proliferating membranes were obtained from six samples each in the intravitreal bevacizumab (IVB) treatment group and the untreated group. The samples were formalin-fixed, paraffin-embedded, cut into sections (5 μm thick), and then dewaxed and rehydrated. After the sections were washed with phosphate-buffered saline (PBS), they were incubated with streptavidin-peroxidase for 10 min, washed, and then blocked in goat serum. Sections were incubated with antibodies against the following antigens at 4° C overnight: transforming growth factor-β1 (TGF-β1, 1:250 dilution, BA0290, Bosterbio), CTGF (1:250 dilution, BA0752, Boster), alpha smooth muscle actin (α-SMA, 1:250 dilution, bs-10196R, Bioss), Collagen Type I Alpha 1 (COL1A1, 1:250 dilution, BA0325, Boster), fibronectin (FN, 1:250 dilution, BA1771, Boster), and vimentin (1:800 dilution, ab128507, Abcam). The sections were then washed with PBS and incubated with biotinylated goat anti-rabbit IgG (1:1000 dilution, ab150077, Abcam) for 1 h at room temperature. The samples were counterstained with DAPI and photographed using a fluorescence microscope. Fluorescence intensity was analyzed using image software and quantitative analysis was performed using ImageJ (Version 1.8.0, http://imagej.nih.gov/ij ).
4
Immunofluorescence Analysis of ECM Proteins
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BEAS-2B cells were plated on cell climbing slices in a 24-well plate. Following the designated treatments, cell climbing slices were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and then incubated with 0.5% triton X-100 for permeabilization. Then, the cells were blocked with 1% BSA for 1 h at room temperature. The primary anti-periostin antibody (1:200, ab152099, Abcam, USA), anti-fibronectin antibody (1:200, JF0582, Huabio, China), and anti-COL1A1 antibody (1:200, BA0325, Boster, China) were used to incubate the cell climbing slices overnight at 4°C. The primary antibody was then removed and fresh PBS was added. After washing with PBS three times, climbing slices were incubated with the secondary antibody goat anti-rabbit IgG (1:500, Abbkine, Hubei, China) and protected from light for 1 h. The nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI) (1:500) for 5 min. After the final washing steps in PBS (3×10 min), the images were captured under an upright fluorescence microscope (Leica, Berlin, Germany).
5
Western Blot Analysis of Cell/Tissue Lysates
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Lysates of tissues or cells were prepared, and western blots were performed43 (link) with the following primary antibodies: anti-collagen I (1:800, BA0325, Boster), anti-fibronectin (1:1000, 15613-1-AP, ProteinTech, IL, USA), anti-LC3 (1:1000, 3868), anti-AMPKα (1:1000, 2532), anti-pAMPKα (Thr172) (1:1000, 50081), anti-ULK1 (1:1000, 8054), anti-pULK1 (Ser555) (1:1000, 5869), anti-pCRTC1 (1:1000, 3359), anti-pCREB(1:1000, 9198s) anti-ATG7 (1:1000, 2631) (all from Cell Signaling Technology), anti-CRTC1(1:1000, PA5-82786, Thermo Fisher Scientific), anti-CREB (1:1000, ab32515, Abcam) or anti-Nox4 (1:500, GTX121929, Gene Tex) antibody, followed by specific secondary antibodies (LICOR, NE, USA). β-actin antibody (1:1000, GTX109639, GeneTex) was used as loading controls.
6
Immunofluorescence Analysis of TNFSF15, Fibronectin, and COL1A1
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BEAS-2B cells were plated on cell climbing slices in a 24-well plate. Following the designated treatments, cell climbing slices were washed with PBS, fixed with 4% paraformaldehyde, and then incubated with 0.5% triton X-100 for permeabilization. Then, the cells were blocked with 1% BSA in PBS for 1 hour at room temperature. The primary anti-TNFSF15 antibody (1:200, DF3053; Affinity, Jiangsu, China), anti-fibronectin antibody (1:200, JF0582; HUABIO), and anti-COL1A1 antibody (1:200, BA0325; Boster) were used to incubate the cell climbing slices overnight at 4°C. The primary antibody was then removed, and fresh PBS was added. After washing with PBS 3 times, climbing slices were incubated with the secondary antibody goat anti-rabbit IgG (1:500; Abbkine Scientific Co., Ltd.) and protected from light for 1 hour. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1:500) for 5 minutes. After the final washing in PBS (3 × 10 minutes), the images were captured under an upright fluorescence microscope (Leica, Berlin, Germany).
7
Protein Expression Analysis by Western Blot
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Protein samples (10 mg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrically transferred onto Immobilon P membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked with 5% skim milkand incubated with primary antibodies including lamin B (1:1000; 66095-1-Ig; ProteinTech Group, Chicago, IL, USA), GAPDH (1:1000; M20005 M; Abmart, China), FLAG (1:1000; F1804; Sigma, St. Louis, MO, USA), hypoxia-inducible factor-1 alpha (HIF-1α) (1:1000; ab51608; Abcam, Cambridge, MA, USA), transforming growth factor (TGF)-β1 (1:1000; ab179695; Abcam), fibronectin (1:1000; ab32419; Abcam), collagen III (1:1000; BA0326; Boster, Wuhan, China), and collagen I (1:1000; BA0325; Boster). Then membranes were incubated with peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using an enhanced chemiluminescence system (Amersham, Buckingshamshire, UK). The density of each band was analyzed by a GS-700 Imaging Densitometer (BIO-RAD, Hercules, CA, USA).
8
Quantitative Western Blot Analysis
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Total cellular proteins were extracted using RIPA and then subjected to western blot experiments for quantification. To carry out western blot experiments, total proteins were segregated using sodium-dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After brief washing, Tris-buffered saline with 1% Tween 20 (TBST) containing 5% bovine serum albumin (BSA) was used for membrane blocking for 1 hour. Then, the membranes were incubated overnight at 4°C with the primary antibodies including anti-TL1A (1:1,000, ab85566; Abcam, Santa Cruz, CA, USA), anti-DR3/LARD antibody (1:1,000, ab189093; Abcam), Anti-COL1A1 antibody (1:1,000, BA0325; Boster, Wuhan, China), anti-fibronectin (1:1,000, JF0582; HUABIO, Hangzhou, China), anti-alpha smooth muscle actin (1:1,000, SY02-64; HUABIO), anti-E-cadherin (1:1,000, PB9561; Boster), anti-E-cadherin (1:1,000, ER0503; HUABIO), and anti-GAPDH (1:1,000, BA2913; Boster). The following day, the membranes were removed with tweezers and then washed in TBST 3 times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (1:3,000, GB23303; Servicebio, Wuhan, China) at room temperature for 1 hour. After washing with TBST again, the membranes were subjected to chemiluminescence by using enhanced chemiluminescence substrate.
9
Histological Analysis of Kidney Tissue
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Commercial kits (Sigma-Aldrich, United States) were used for Masson or HE staining of kidney tissue according to the manufacturer’s protocol. Masson staining and immunostaining intensity were scored by photograph, randomly selected under 200 magnified visual field with a light microscope. According to the degrees of tubule degeneration and necrosis, tubule atrophy, inflammatory cell infiltration, and fibrosis, 0, 1, 2, and 3 were scored. The collagen-blue area representing the lesion area after Masson staining was calculated. The staining area was measured by the average optical density of the immunostaining intensity score. The ratios of FABP4-positive area to total area in kidney in glomeruli at a magnification of ×400 and in the cortical tubulointerstitium except glomeruli and vascular lumen at a magnification of ×200 were calculated as FABP4 positive in glomerular and FABP4 positive in interstitial, respectively.
Immunohistochemical staining was performed on 4-μm kidney sections as previously described. After antigen retrieval, sections were incubated with the primary antibody against α-SMA (ab5694; Abcam, United States), Col-1 (BA0325; Bosterbio, United States), FN (BA1772; Bosterbio), FABP4 (ET1703-98, HuaBio, China), F4/80 (RT1212; HuaBio), or CD68 (ab955; Abcam), respectively, and then detected by the En Vision/HRP Kit (Dako, Carpinteria, CA, United States).
Immunohistochemical staining was performed on 4-μm kidney sections as previously described. After antigen retrieval, sections were incubated with the primary antibody against α-SMA (ab5694; Abcam, United States), Col-1 (BA0325; Bosterbio, United States), FN (BA1772; Bosterbio), FABP4 (ET1703-98, HuaBio, China), F4/80 (RT1212; HuaBio), or CD68 (ab955; Abcam), respectively, and then detected by the En Vision/HRP Kit (Dako, Carpinteria, CA, United States).
10
Collagen I and III Immunohistochemistry
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The paraffin heart sections (5 μm thickness) were treated with 3% hydrogen peroxide for 5 min. The sections then were incubated with the primary antibodies: anti-collagen I (1:200 dilution; BA0325, Boster, Wuhan, China) and anti-collagen III (1:200 dilution; BA0326, Boster, Wuhan, China) at 4°C overnight. After being washed, the paraffin sections were incubated with goat anti-rabbit immunoglobulin G biotinylated secondary antibody (1:200 dilution; SA1022, Boster, Wuhan, China) for 1 h at room temperature, and stained using a DAB detection kit (AR1000, Boster, Wuhan, China).
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