The proteins, extracted from human or mice skin samples as well as mice skin fibroblasts, were separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the membranes were immerged into 5% milk without fat to block for 2 hours. Next, they were incubated with primary antibodies including anti‐SIRT3, caspase 3, MLKL, p‐MLKL, RIPK1 (1:1000, Cell Signaling Technology), anti‐RIPK3 (1:1000, Novusbio), anti‐α‐SMA (1:1000, Bosterbio), anti‐GAPDH (1:5000, Sigma‐Aldrich) and anti‐β‐tubulin (1:3000, CMCTAG) at 4°C for more than 12 hours. Horseradish peroxidase (HRP)‐conjugated IgG (Beyotime) or enhanced chemiluminescence (ECL, Thermo Fisher Scientific Inc) was applied to visualize the protein bands.
Anti α sma
Manufactured by Boster Bio
Sourced in China, United States
Anti-α-SMA is a monoclonal antibody that specifically recognizes the alpha-smooth muscle actin (α-SMA) protein. α-SMA is a cytoskeletal protein expressed in smooth muscle cells and myofibroblasts. This antibody can be used in various applications, such as immunohistochemistry and Western blotting, to detect and quantify the presence of α-SMA in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Proteintech (4 mentions)
Anti vwf, by Santa Cruz Biotechnology (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
P mlkl, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti cd31, by Abcam (2 mentions)
Rapamycin, by Merck Group (2 mentions)
Anti cd31, by Santa Cruz Biotechnology (2 mentions)
Anti ulk1, by Proteintech (2 mentions)
Bafilomycin a1, by Merck Group (2 mentions)
Anti β actin, by Proteintech (2 mentions)
Anti ampk, by Proteintech (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti ripk3, by Novus Biologicals (1 mentions)
Horseradish peroxidase hrp conjugated igg, by Beyotime (1 mentions)
Ripk1, by Cell Signaling Technology (1 mentions)
Hoechst 33342 staining, by Beyotime (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Cyanine3 (cy3), by Beyotime (1 mentions)
Ripk3, by Cell Signaling Technology (1 mentions)
16 protocols using anti α sma
1
Protein Expression Analysis in Skin Samples
2
Histological Analysis of Kidney Tissues
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After embedding kidney tissues into paraffin, the 3 μm sections were used for hematoxylin and eosin (HE) staining according to the standard protocol. Histopathological changes were scored as follows: 0, no damage; 1, <25%; 2, 25-50%; 3, 50-75%; and 4, >75%. IHC staining was performed as previously described [8 (link)]. The primary antibody for IHC staining was anti-α-SMA (1 : 100, Boster, China). 4 random fields per section were selected for the analysis of semiquantification by Image-Pro Plus 6.0 software.
The Hoechst 33342 staining (Beyotime, Shanghai, China) was used to identify the morphology of apoptotic nuclei. The sections were deparaffinized and then exposed to the Hoechst 33342 solution for 5 min at 37°C in the dark. The positive nuclei were observed with a fluorescence microscope (Nikon Eclipse80i, Japan) at 200x magnification.
The Hoechst 33342 staining (Beyotime, Shanghai, China) was used to identify the morphology of apoptotic nuclei. The sections were deparaffinized and then exposed to the Hoechst 33342 solution for 5 min at 37°C in the dark. The positive nuclei were observed with a fluorescence microscope (Nikon Eclipse80i, Japan) at 200x magnification.
3
Immunofluorescence Analysis of Cardiac Fibroblasts
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After treatment and fixation, cardiac fibroblasts were incubated with blocking solution at room temperature. Then, diluted primary anti-α-SMA (1:100), collagen I (1:200), and collagen III (1:200) (Boster Biological Technology, Dublin, CA, USA); ki67 (1:100), PCNA (1:200) (ABclonal Technology, Wuhan, China); and the DRP1, OPA1, or RIPK3 (1:50, Cell Signaling Technology, Danvers, MA, USA) antibody were added and incubated overnight at 4 °C. After washing, the cells were incubated by IgG conjugated with Alexa Fluor 488 or Cy3 (1:500, Beyotime, Shanghai, China) without light at room temperature for 2 h followed by DAPI staining for 15 s. The cells were observed and photographed with a laser confocal microscope. The protein expression, which is considered as the fluorescence intensity, was quantified using ImageJ software.
4
Cardiac Cell Phenotyping Using Immunofluorescence
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Heart sections were stained with anti-MLC2V (Proteintech; 1:50), anti-α-SMA (BOSTER; 1:50) and anti-CD31 (Abcam, 1:50) at 4 °C overnight. The samples were washed with PBS and then incubated with secondary antibodies at room temperature for 1 h. DAPI was used for nuclear staining. The immunofluorescence-stained sections were imaged with a Zeiss confocal microscope.
5
Immunofluorescence Assay for Kir2.1, α-SMA, Collagen I, and III
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After culturing and incubation for 24 h, the cells were washed with PBS three times (2 min each) and then fixed with paraformaldehyde for 10 min. After being washed with PBS three times (2 min each), the cell membrane was permeabilized with Triton X-100 for 1–3 min and then washed again with PBS three times (2 min each). After blocking with 10% BSA for 30 min, the antibodies were incubated with anti-Kir2.1 (1:100; Abcam), anti-α-SMA (1:100; Boster), anti-collagen I (1:100; Boster), or anti-collagen III (1:100; Boster) antibodies at 4°C overnight. After 30 min of rewarming the next day, the corresponding fluorescein-labelled antibody was added and incubated for 2 h at 37°C. PBS was used to wash the cells three times (10 min each). DAPI was used to stain the nuclei for 10 min, and then cells were observed under a fluorescence microscope (Solarbio, Beijing, China), and images were captured. ImageJ software (NIH, Bethesda, USA) was used for fluorescence image analysis.
6
Naringenin Mitigates Diabetic Nephropathy
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Naringenin (Nar) (≥ 95%, N5893), streptozotocin (STZ) were purchased from Sigma (Sigma Aldrich, United States). Anti-PCNA (1:1000, ab29) and anti-OPN (1:500, ab63856) were purchased from Abcam. Anti-MMP2 (1:1000, bs-4605R) and anti-MMP9 (1:1000, bsm-54040R) were obtained from Bioss Biotechnology Co., LTD. (Beijing, China). Anti-α-SMA (1:1000, BM0002), anti-VEGFA (1:1000, BA0407) and anti-VEGFR2/KDR (1:1000, A00901-3) were purchased from Boster Biological Technology Co. Ltd. (Shanghai, China). Anti-CREB5 (1:1000, G420) was purchased from Santa cruz. The following antibodies: PI3K (1:1000, 4249), p-PI3K (1:500, 17366), Akt-pan (1:1000, 4685), p-Akt (Ser 473) (1:1000, 4060), Src (1:1000, 36D10), and p-Src (1:1000, D49G4) were obtained from CST. EdU Imaging kit was purchased from APE × Bio (United States). Trypsin, Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO.
7
Detailed Procedures for Cellular Senescence Assessment
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The reagents included carbon tetrachloride (Sigma‐Aldrich, 56‐23‐5), 30% H2O2 (Hydrogen peroxide 30%, Sigma‐Aldrich, 1.07298), selisistat (EX‐527, MedChemExpress, 49843‐98‐3), resveratrol (SRT501, MedChemExpress, 501‐36‐0), DAPI (Sigma‐Aldrich, D9542), Alexa Fluor™ 647 Phalloidin (Thermo, A22287), protease cocktails inhibitor (Beyotime, P1005) and PMSF (Phenylmethanesulphonyl fluoride, Beyotime, ST506).
The primary antibodies included anti‐α‐SMA (Boster, BM0002), anti‐vWF (Santa Cruz, SC‐365712), anti‐CD32b (ZEN‐bioscience, 382560), anti‐CD31 (PECAM‐1, Santa Cruz, sc‐18916), anti‐CD31 (Abcam, ab33858), anti‐NOX2 (Proteintech, 19013‐1‐AP), anti‐NOX4 (Proteintech, 14347‐1‐AP), anti‐Lamin A/C (Cell Signaling Technology, 4777S), anti‐Lamin B1 (Proteintech, 66095‐1‐Ig), anti‐progerin (Santa Cruz, sc‐81611), anti‐p53 (Abcam, ab131442), anti‐p53 (acetyl K381; Abcam, ab61241), anti‐SIRT1 (Abcam, ab110304), anti‐Histone H3 (Proteintech, 17168‐1‐AP) and anti‐GAPDH (Proteintech, 60004‐1). HRP‐conjugated Affinipure Goat Anti‐Mouse IgG (H + L; Proteintech, SA00001‐1), HRP‐conjugated Affinipure Goat Anti‐Rabbit IgG (H + L; Proteintech, SA00001‐2), FITC‐labelled goat anti‐rabbit IgG (H + L; Beyotime, a0562) and Cy3‐labelled goat anti‐mouse IgG (H + L; Beyotime, a0521) were used for secondary antibodies.
The primary antibodies included anti‐α‐SMA (Boster, BM0002), anti‐vWF (Santa Cruz, SC‐365712), anti‐CD32b (ZEN‐bioscience, 382560), anti‐CD31 (PECAM‐1, Santa Cruz, sc‐18916), anti‐CD31 (Abcam, ab33858), anti‐NOX2 (Proteintech, 19013‐1‐AP), anti‐NOX4 (Proteintech, 14347‐1‐AP), anti‐Lamin A/C (Cell Signaling Technology, 4777S), anti‐Lamin B1 (Proteintech, 66095‐1‐Ig), anti‐progerin (Santa Cruz, sc‐81611), anti‐p53 (Abcam, ab131442), anti‐p53 (acetyl K381; Abcam, ab61241), anti‐SIRT1 (Abcam, ab110304), anti‐Histone H3 (Proteintech, 17168‐1‐AP) and anti‐GAPDH (Proteintech, 60004‐1). HRP‐conjugated Affinipure Goat Anti‐Mouse IgG (H + L; Proteintech, SA00001‐1), HRP‐conjugated Affinipure Goat Anti‐Rabbit IgG (H + L; Proteintech, SA00001‐2), FITC‐labelled goat anti‐rabbit IgG (H + L; Beyotime, a0562) and Cy3‐labelled goat anti‐mouse IgG (H + L; Beyotime, a0521) were used for secondary antibodies.
8
Liver Protein Analysis by Western Blot
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Total protein samples were extracted from liver tissue using RIPA buffer (G2002, Servicebio), and the protein concentration was determined using a BCA protein assay kit (G2026, Servicebio). Protein samples were resolved using 10% SDS-PAGE and then transferred onto a PVDF membrane (IPVH00010, Millipore, Danvers, MA, USA). Membranes were blocked and incubated with primary antibodies overnight at 4°C. A total of 8 commercial antibodies were used for Western blotting, including anti-α-SMA (1 : 1000, Boster, USA), anti-TGFβ1 (transforming growth factor beta, 1 : 1000, Abcam), anti-collagen III (1 : 1000, ab6310, Abcam), anti-VEGF (1 : 1000, ab46154, Abcam), anti-CYP3A1 (1 : 1000, ab22724, Abcam), anti-ALB (albumin, 1 : 1000, Abcam), anti-VCAM-1 (1 : 1000, Abcam), and β-actin (1 : 1000, Servicebio). Membranes were incubated at room temperature with an appropriate secondary antibody, and proteins were detected using an enhanced chemiluminescence method. Protein expression was normalized to the expression of β-actin which was used as an internal control.
9
Protein Expression Analysis in Skin Fibroblasts
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The proteins from skin fibroblasts were separated by the sodium dodecyl sulphate- (SDS-) polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride (PVDF, Millipore, Billerica, MA, USA) membrane. The membranes were blocked by 5% milk without fat for 2 h, then incubated with anti-α-SMA (1 : 2000; Bosterbio, Wuhan, China), anti-PCNA (1 : 2000; ABclonal, Wuhan, China), anti-receptor interacting protein kinase 1 (RIPK1), RIPK3, anti-mixed lineage kinase domain like protein (MLKL), p-MLKL (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH (1 : 3000; Sigma-Aldrich, St. Louis, MO, USA), anti-β-tubulin (1 : 3000; CMCTAG, Milwaukee, WI, USA), or anti-β-actin (1 : 5000; ABclonal, Wuhan, China) antibodies at 4°C overnight. Next, the secondary antibody (1 : 5000; Beyotime, Shanghai, china) was incubated for 2 h at room temperature. The protein bands were visualized on the membrane with enhanced chemiluminescence (ECL, Thermo Fisher Scientific Inc., Rockford, IL, USA) solution.
10
Autophagy Regulation in Endothelial Cells
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The reagents used included 3MA (Sigma-Aldrich, S2767), rapamycin (Sigma-Aldrich, S1039), bafilomycin A1 (Sigma-Aldrich, SML1661), VEGF (PeproTech, 100-20A), N-Nitro-L-Arginine Methyl Ester (L-NAME, Sigma-Aldrich, N5751), Diethylenetriamine/nitric oxide adduct (2,2′-(Hydroxynitrosohydrazono)bis-ethanimine DETA/NO, NO donor, Sigma-Aldrich, D185).
The antibodies used included anti-Col I (Proteintech, 14695-1-AP), anti-α-SMA (Boster, BM0002), anti-vWF (Santa Cruz, SC-365712), anti-CD31 (Santa Cruz, SC-46694), anti-Cav-1 (Abcam, ab17052), anti-Cav-1 (Abcam, ab2910), anti-LC3 (Abcam, ab48394), anti-p-VASP (Ser157) (CST, 84519), anti-VASP (CST, 3132S), anti-p-eNOS (Ser1177) (CST, 9570), anti-eNOS (Abclonal, A1548), anti-p-PI3K (Tyr458) (CST, 4228), anti-PI3K (CST, 4249), anti-p-AKT (Ser473) (CST, 4060), anti-AKT (CST, 2938), anti-p-MTOR (Ser2448) (CST, 5536), anti-MTOR (CST, 2983), anti-GLUT3 (Abcam, ab41525), anti-AMPK (Proteintech, 10929-2-AP), anti-ULK1 (Proteintech, 20986-1-AP), anti-GAPDH (Proteintech, 60004-1), and anti-β-actin (Proteintech, 60008-1). DAPI (Sigma-Aldrich, D9542), FITC-labeled goat anti-rabbit IgG (H+L) (Beyotime, a0562), Cy3-labeled goat anti-mouse IgG (H+L) (Beyotime, a0521), and phallotoxins (Thermo, F432) were also used.
The antibodies used included anti-Col I (Proteintech, 14695-1-AP), anti-α-SMA (Boster, BM0002), anti-vWF (Santa Cruz, SC-365712), anti-CD31 (Santa Cruz, SC-46694), anti-Cav-1 (Abcam, ab17052), anti-Cav-1 (Abcam, ab2910), anti-LC3 (Abcam, ab48394), anti-p-VASP (Ser157) (CST, 84519), anti-VASP (CST, 3132S), anti-p-eNOS (Ser1177) (CST, 9570), anti-eNOS (Abclonal, A1548), anti-p-PI3K (Tyr458) (CST, 4228), anti-PI3K (CST, 4249), anti-p-AKT (Ser473) (CST, 4060), anti-AKT (CST, 2938), anti-p-MTOR (Ser2448) (CST, 5536), anti-MTOR (CST, 2983), anti-GLUT3 (Abcam, ab41525), anti-AMPK (Proteintech, 10929-2-AP), anti-ULK1 (Proteintech, 20986-1-AP), anti-GAPDH (Proteintech, 60004-1), and anti-β-actin (Proteintech, 60008-1). DAPI (Sigma-Aldrich, D9542), FITC-labeled goat anti-rabbit IgG (H+L) (Beyotime, a0562), Cy3-labeled goat anti-mouse IgG (H+L) (Beyotime, a0521), and phallotoxins (Thermo, F432) were also used.
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