Tissues or cells were collected and lysed in radioimmunoprecipitation assay (RIPA, Solarbio, R0020) buffer and phenylmethylsulfonyl fluoride (PMSF, Solarbio, P0100) (RIPA buffer: PMSF = 100:1). The protein concentration was measured by bicinchoninic acid protein assay kit (Coolaber, SK1070). After separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred to polyvinylidenefluoride membranes. The protein was sealed for 2 h with 5% skim milk and incubated overnight at 4°C with antibodies: anti-collagen I (Affinity, AF7001), anti-collagen III (Affinity, AF0136), anti-vimentin (Affinity, AF7013), anti-α-SMA (Affinity, AF1032), anti-FAP1 (Cell Signaling Technology, 66562S), anti-S100A4 (Cell Signaling Technology, 13018S), anti-Myo1c (Abcam, ab194828), anti-YAP1 (Cell Signaling Technology, 14074S), anti-phospho-YAP1 (Cell Signaling Technology, 53749S), anti-F-actin (Abcam, ab130935), anti-GAPDH (Affinity, AF7021). 1×Tris buffered saline Tween was used to wash the membranes for three times. Then membranes were incubated for 1 h with secondary antibodies at room temperature. The expression of proteins was detected by enhanced chemiluminescence reagent kit (SparkJade, ED0015-B).
Anti s100a4
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom
Anti-S100A4 is a primary antibody that specifically binds to the S100A4 protein. S100A4 is a calcium-binding protein involved in various cellular processes. This antibody can be used to detect and study the expression and localization of S100A4 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid protein assay kit, by Coolaber (1 mentions)
Anti collagen 3, by Affinity Biosciences (1 mentions)
Anti fap1, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Anti yap1, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Affinity Biosciences (1 mentions)
Anti collagen 1, by Affinity Biosciences (1 mentions)
Anti f actin, by Abcam (1 mentions)
Anti vimentin, by Affinity Biosciences (1 mentions)
Anti α sma, by Affinity Biosciences (1 mentions)
Anti phospho yap1, by Cell Signaling Technology (1 mentions)
R0020, by Solarbio (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Anti osx antibody, by Abcam (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
General chemicals, by Merck Group (1 mentions)
Short interfering rna sirna duplexes, by RiboBio (1 mentions)
Anti cd44, by Proteintech (1 mentions)
7 protocols using anti s100a4
1
Protein Expression Analysis in Tissue Samples
2
S100A4 Signaling in Cell Regulation
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Roswell Park Memorial Institute (RPMI)‐1640 medium, Dulbecco's modified Eagle's medium (DMEM), DMEM/F12, Opti‐MEM Reduced Serum Medium, and fetal bovine serum (FBS) were purchased from Gibco (Invitrogen, Carlsbad, CA, USA). General chemicals were purchased from Sigma (St. Louis, MO, USA) unless specifically mentioned. Short interfering RNA (siRNA) duplexes targeting the human S100A4 gene and siRNA duplexes with nonspecific sequences were designed and synthesized by RiboBio (Guangzhou, China). Anti‐OSX antibodies were purchased from Abcam (Cambridge, MA, USA). Anti‐S100A4 and anti‐β‐catenin antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti‐β‐actin, anti‐CD34, and horseradish peroxidase‐conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti‐CD44, anti‐VEGF, and anti‐CD31 antibodies were obtained from Proteintech (Chicago, IL, USA).
3
Immunoblot Analysis of NF-κB Pathway
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Immunoblots were performed as previously described [46 (link)] with the following primary antibodies: anti-GAPDH (sc-32,233, Santa Cruz), anti-p100/p52 (4882 or 37,359 S, Cell Signaling), anti-p105/p50 (D4P4D, Cell Signaling), anti-lamin (4777 S, Cell Signaling) anti-p65 (D14E12, Cell Signaling), anti-RelB (10,544 S, Cell Signaling), anti-IKKα (2682 S, Cell Signaling), anti-NIK (4994 S, Cell Signaling), anti-HA (sc-7392, Santa Cruz), anti-Actin (sc-8432, Santa Cruz), anti-CYPA (sc-134,310, Santa Cruz), anti-S100A4 (13,018 S, Cell Signaling), anti-Histone H3 (sc-517,576, Santa Cruz), anti-STAT3 (sc-482 X, Santa Cruz), and anti-phospho-STAT3 (sc-8059, Santa Cruz). All immunoblots are representative of at least two separate experiments and representative images displayed.
4
S100A4 Protein Expression Analysis
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The cultured EOC cells were harvested and lysed in RIPA buffer (Amresco N653) at 4°C for 40 min and then separated by electrophoresis, incubated with anti-S100A4 and anti-actin (Cell Signaling 3285, Shanghai, China).
5
Aortic Tissue Characterization in Aneurysm
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Normal aortic tissue was obtained from patients undergoing coronary artery bypass grafting (n = 11; male = 6, female = 5). Aneurysmal aortas (n = 12 ascending thoracic aortic aneurysm [aTAA]; n = 13 descending thoracic aortic aneurysm [dTAA]; n = 13 abdominal aortic aneurysm [AAA]) were taken from patients undergoing elective open AA repair. Aortas with known collagen vascular disease, genetic aortopahy, or bicuspid aortic valve were excluded.
Aortic tissue specimens were explanted, placed in 4% paraformaldehyde overnight, and embedded in paraffin. Immunohistochemistry was performed and digitally analyzed, as previously described. Antibodies for immunohistochemical staining were anti-Snail (1:300, Abcam), anti-Slug (1:600, Abcam), anti-S100A4 (1:500, Cell Signaling Technologies), and anti-VWF (1:1500, Abcam).
Aortic tissue specimens were explanted, placed in 4% paraformaldehyde overnight, and embedded in paraffin. Immunohistochemistry was performed and digitally analyzed, as previously described. Antibodies for immunohistochemical staining were anti-Snail (1:300, Abcam), anti-Slug (1:600, Abcam), anti-S100A4 (1:500, Cell Signaling Technologies), and anti-VWF (1:1500, Abcam).
6
Comparative Western Blot Analysis of ECM Markers
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Western blotting was conducted as described previously, with minor modifications [29 (link)], using the following primary antibodies: anti-α-SMA (1:1,000; Sigma-Aldrich, St. Louis, MO, USA), anti-fibronectin (FN) (1:1,000; Abcam, Cambridge, UK), anti-Col I (1:1,000; Abcam), anti-YAP (1:1,00, cat. no. sc-101199; Santa Cruz Biotechnology, Dallas, TX, USA), anti-TAZ (1:1,000, cat. no. 8418; Cell Signaling Technology), and anti-S100A4 (1:1,000, cat. no. ab124805; Abcam). The secondary antibodies included horseradish peroxidase (HRP)-linked anti-rabbit (1:2,000; Cell Signaling Technology), HRP-linked anti-mouse (1:2,000; Cell Signaling Technology), and mouse anti-mouse (1:20,000; GE Healthcare, Chicago, IL, USA). The experiments were conducted in triplicate.
7
Immunohistochemistry Staining Protocol
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Immunohistochemistry was performed as described previously (Fu et al., 2019 (link)). In short, paraffin sections (5 μm) were deparaffined, rehydrated, and antigen retrieved by boiling in 10 mM citrate buffer for 10 min. Endogenous horseradish peroxidase (HRP) activity was inhibited with 3% H2O2 solution in methanol. After washing for three times with PBS, the sections were incubated at 37℃ for 1 hr in 10% horse serum for blocking, incubated overnight in each primary antibody at 4 °C. The primary antibodies used in this study included anti-TNC (1:200, ab108930, Abcam, Cambridge, UK) and anti-S100A4 (1:200, 13018 S, Cell Signaling Technology). After washing, the sections were incubated with biotinylated rabbit anti-goat IgG antibody (1:200, Zhongshan Golden Bridge, Beijing, China) and streptavidin-HRP complex (1:200, Zhongshan Golden Bridge). According to the manufacturer’s protocol, the positive signals were visualized using DAB Horseradish Peroxidase Color Development Kit (Zhongshan Golden Bridge). The nuclei were counter-stained with hematoxylin. Each experiment was repeated at least three times.
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