Antibodies used for Western blotting and immunofluorescence were as follows: anti-α-SMA (ab5694; Abcam, Cambridge, England), anti-collagen Type I (600-401-103-0.1; Rockland, Inc., PA, USA), anti-fibrinogen (ab23750, Abcam), anti-Periostin (ab14041, Abcam), anti-Smad2 (#3103S; Cell Signaling, Beverly, MA, USA), anti-phosphorylated Smad2 (#3101S; Cell Signaling), anti-Stat3 (#9139S; Cell Signaling), and anti-phosphorylated Stat3 (#9145S, Cell Signaling). Antibodies for the immunohistochemistry experiments were as follows: anti-E-cadherin mAb (M3612; Dako, Carpinteria, CA, USA), anti-α-SMA (ab5694; Abcam), and anti-Ki67 (M7240; Dako). Transforming growth factor (TGF)-β1 was purchased from R&D Systems (240-B; Minneapolis, MN, USA). For in vitro studies, PFD was provided by Shionogi & Co., Ltd. (Osaka, Japan) and dissolved in DMSO to a concentration of 50 μg/mL. For in vivo studies, Pirespa tablets were purchased from Shionogi & Co. and dissolved in DMSO, using one-fifth of the final volume of the total solvent, after crushing using a micro smash (TOMY SEIKO CO., LTD, Tokyo, Japan). Sterile normal saline at four-fifths of the final total volume was then added to bring PFD to a final concentration of 40 mg/mL.
Anti α sma
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Japan
Anti-α-SMA is an antibody that specifically binds to alpha-smooth muscle actin (α-SMA), a cytoskeletal protein expressed in smooth muscle cells. This antibody can be used in various immunoassays and research applications to detect and quantify α-SMA levels.
Automatically generated - may contain errors
Lab products found in correlation
Anti collagen 1, by Abcam (30 mentions)
Anti e cadherin, by Abcam (25 mentions)
Anti vimentin, by Abcam (23 mentions)
Anti fibronectin, by Abcam (22 mentions)
Anti β actin, by Merck Group (20 mentions)
Anti gapdh, by Abcam (16 mentions)
Anti cd31, by Abcam (15 mentions)
Anti col1, by Abcam (13 mentions)
Pvdf membrane, by Merck Group (13 mentions)
Ripa buffer, by Thermo Fisher Scientific (12 mentions)
Anti tgf β1, by Abcam (10 mentions)
Anti β actin, by Abcam (10 mentions)
Anti cd68, by Abcam (10 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Anti e cadherin, by Cell Signaling Technology (9 mentions)
Anti vimentin, by Cell Signaling Technology (9 mentions)
Anti gapdh, by Cell Signaling Technology (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (9 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (9 mentions)
Lsm 710 confocal microscope, by Zeiss (9 mentions)
354 protocols using anti α sma
1
Antibody-based Analysis of Fibrosis Markers
2
Investigating Fibrosis Markers in Mice
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Bleomycin, DNase I, paraformaldehyde, mouse IgG, and mouse serum were purchased from Sigma-Aldrich (St. Louis, MO, USA), and simvastatin, obtained from the Cayman Chemical Co. (Ann Arbor, MI, USA), was used as indicated.). Dispase was obtained from Corning Incorporated (Corning, NY, USA). Antibodies for western blotting were as follows: anti-CD36, anti-MMR (Cayman Chemical Co), anti-α- SMA, anti-fibronectin (Abcam, Cambrige, MA, USA), anti-type 1 collagen α2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-α-SMA (Abcam), and anti-β-actin (Sigma-Aldrich).
3
Western Blot Analysis of miRNA-144 Transfected Cells
4
Immunohistochemical Characterization of Hepatic Cells
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Optimal cutting temperature (OCT) compound embedded liver biopsy cryosections and paraffin-embedded sections (both 5 μm) were incubated with anti-NKp46 (Clone 195314, R&D Systems), anti-SMA-α (Rabbit polyclonal, Abcam) or TGF-β (Rabbit polyclonal, Abcam), respectively, according to our previously described protocols26 (link)38 (link). Double staining was performed using mouse anti-NKp46 and anti-SMA-α to evaluate the co-location of hepatic NK cells and HSCs. High-powered fields (hpf, 400×) were used to count positive cells. Positive cells were counted in three different fields by two independent observers.
5
Western Blot Protein Quantification and Analysis
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The cells were collected and lysed, and sample proteins were quantitated using pierce BCA protein assay (Thermo Fisher Scientific, Rockford, USA). Equal amounts of proteins were separated by SDS-PAGE and transferred to PVDF membranes. Primary antibodies and dilutions used for Western blotting included the following: anti-P27 (1 : 1,000 dilution, Abcam, Boston, MA, USA), antivimentin (1 : 1000 dilution, Abcam), and mouse anti-GAPDH (1 : 4,000 dilution, Abcam); anti-α-SMA (1 : 1,000 dilution, Abcam), anti-β-actin (1 : 2,000 dilution, Cell Signaling, Beverly, MA, USA), anti-MTORC-p (1 : 1000 dilution, Abcam), and anti-Rictor (1 : 2000 dilution, Abcam). Secondary antibody (horseradish peroxidase-conjugated immuno-pure anti-IgG; H + L) was used at a dilution of 1 : 5,000. The blots were developed with chemiluminescence reagent ECL kit (Beit Haemek, Israel).
6
Western Blot Analysis of Vascular Smooth Muscle Cell Proteins
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Protein was isolated from VSMCs or aortic tissues using a RIPA lysis buffer kit (Santa Cruz Biotechnology, Inc., USA). The protein content was determined in the supernatants using a Bio-Rad protein assay (Bio-Rad Laboratories, Inc., USA). Protein lysates (30 μg/sample) were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF, Millipore Corp., USA) membranes. The membranes were blocked in 5% fat-free milk overnight at 4°C. Membranes were then probed with the following primary antibodies purchased from Abcam (Cambridge, USA): anti-α-SMA (cat. no. ab5694, 1:1,000), anti-OPN (cat. no. ab8448, 1:1,000), anti-MMP-2 (cat. no. ab92536, 1:1,000), anti-MMP-9 (cat.no. ab38898, 1:1,000), and anti-TIMP-1 (cat. no. ab38978, 1:1,000) and incubated overnight at 4°C. Subsequently, protein bands were detected by incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG, cat. no. ab6789; 1:2,000; Abcam) at room temperature for 1 h. Signals were detected using an enhanced chemiluminescence kit (ECL kit, Wuhan Boster Biotechnology Co., Ltd, China) and exposed to Kodak X-OMAT film (Kodak, Rochester, USA). The band intensity was quantified with the Quantity One software. Each experiment was performed at least three times. GAPDH served as the loading control.
7
Molecular Mechanisms of TGF-β1-Mediated Apoptosis
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Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA), emodin was obtained from Shanghai future industry Limited by Share Ltd (Shanghai, China) and BLM was acquired from Nippon Kayaku (Tokyo, Japan). The primary antibodies described in the study include: anti-E-cadherin, anti-vimentin, anti-cleaved caspase-3, anti-phospho-Smad2, anti-phospho-Smad3, anti-Smad2, anti-Smad3, anti-phospho-Erk1/2 and anti-Erk1/2 (Cell Signaling Technology, CA, USA); anti-caspase-3, anti-Bax (Santa Cruz Biotechnology, CA, USA); anti-fibronectin (Proteintech, Chicago, USA); anti-caspase-8, anti-Bcl-2 (Absci, MD, USA); anti-TGF-β1, anti-FSP-1, anti-α-SMA (Abcam, USA); and anti-GAPDH (Beyotime Institute of Biotechnology, Haimen, China). Other reagents were obtained from Beyotime Institute of Biotechnology unless otherwise indicated.
8
Western Blot Analysis of Metabolic Proteins
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Western blot was performed as previously described [16 (link)]. After incubation with the secondary antibody, the proteins were detected by chemiluminescence. Primary antibodies used in this study were anti-HMGCS2 (for WB) (1:2000; Abcam ab137043), anti-α-SMA (1:2000; Abcam, ab7817), anti-Collagen1 (1:1000; Cell Signaling Technology, #72026), anti-Fibronectin (1:2000; Cell Signaling Technology, #26836), anti-PPARα (1:1000; Affinity, AF5301), anti-CPT1A (1:1000; Affinity, AF5301), anti-CPT2 (1:1000; Affinity, AF5301), and anti-GAPDH (1:4000; Beyotime, China).
9
Cell Lysis and Western Blot Analysis
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Whole-cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described24 (link). Nuclear lysates were prepared with the NE-PER Kit (Pierce) following manufacturer’s recommendation. Western blot analyses were performed with anti-MKL1 (Santa Cruz, sc-32909), anti-collagen type I (Rockland, 600-401-103), anti-α-SMA (Abcam, ab5694), anti-STAT3 (Cell Signaling Technology, 9132), anti-TWIST1 (Abcam, ab50887), anti-VE-Cadherin (Cell Signaling Technology, 2158), anti-PECAM1 (Proteintech, 11265-1), anti-α-tubulin (Sigma, T5168), anti-Lamin B (Santa Cruz, sc-6216), and anti-β-actin (Sigma, A2228) antibodies. All experiments were repeated three times.
10
Protein Extraction and Western Blot Analysis
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Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mMNaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described (Fan et al., 2020 (link)). Nuclear proteins were extracted using the NE-PER Kit (Pierce) following manufacturer’s recommendation (Mao et al., 2020a (link)). Western blot analyses were performed with anti-MRTF-A (Santa Cruz, sc-32909), anti-SRF (Cell Signaling Technology, 5147), anti-α-tubulin (Sigma, T6074), anti-Lamin A/C (Proteintech, 10298-1), anti-α-SMA (Abcam, ab5694), and anti-β-actin (Sigma, A1978). For densitometrical quantification, densities of target proteins were normalized to those of β-actin as previously described (Lv et al., 2020 (link); Wu et al., 2020 (link)). Data are expressed as relative protein levels compared to the control group which is arbitrarily set as 1.
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