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Anti β actin

Manufactured by Abcepta
Sourced in United States

Anti-β-actin is a primary antibody used for the detection and quantification of β-actin, a ubiquitous and highly conserved cytoskeletal protein. This antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and ELISA, to measure the expression levels of β-actin in biological samples.

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7 protocols using anti β actin

1

Quantitative Western Blot Analysis Protocol

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Western blotting was carried out as described previously (Lin et al., 2019 (link); Wu et al., 2019 (link)). Briefly, the proteins from each cell layer extract were quantified using the BCA Protein kit (Beyotime Biotechnology, Shanghai, China). Protein extracts were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). Membranes were blocked with 5% non-fat milk in Tris–buffered saline for 1 h at room temperature. The membranes were incubated with primary antibodies, including anti-RAP2a (1:2,000; catalog no. NBP2-24574; Novusbio), DNMT3a (1:500; catalog no. 2160; Cell Signaling Technology), and anti-β-actin (1:3,000; catalog no. AP53385; Abgent) at 4°C overnight, followed by incubation with HRP-conjugated goat anti-rabbit (1:5,000; catalog no. sc-2004; Santa Cruz Biotechnology; RRID:AB_631746) or HRP-conjugated goat anti-mouse (1:5,000; sc-2005; Santa Cruz Biotechnology; RRID:AB_631746) secondary antibodies at 37°C for 1 h. The immunoreactive bands were visualized by using the ECL Plus Western Blot Detection kit (Amersham Biosciences United Kingdom), and densitometric quantification of band intensity from three independent experiments was carried out with the Image-Pro Plus 6.0 software. The relative expression level of the target protein was normalized to the intensity of the β-actin band.
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2

Thrombi Processing and Antibody Detection

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Processing of thrombi and detection of primary antibodies is described in the Online Supplementary Methods. Membranes were probed with 2 μg/mL anti-Ly6G (BioXCell, West Lebanon, NH, USA), a 1,000-fold dilution of anti-β-actin (Abgent, San Diego, CA, USA), a 2,000-fold dilution of anti-PAD4 (Abcam), 1 μg/mL anti-H3Cit (Abcam) or 0.5 μg/mL anti-histone H3 (Abcam) primary antibody.
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3

Immunoblot Analysis of NLRP3 Inflammasome

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HCASMC were homogenized in RIPA buffer (Cell Signaling, USA) containing protease inhibitor cocktail HALT (ThermoFisher, USA). For immunoblot analysis 20–30 µg of protein lysate was resolved on Any kD Mini-PROTEAN TGX Precast polyacrylamide gels (Biorad, Hercules, CA, USA), transferred to nitrocellulose membrane, blocked in 5% Blotting-grade blocker (Biorad) and incubated with appropriate primary antibodies. Anti-IL1β (1:1000, Abcam), anti-caspase-1 p20 Bally-1 (Adipogen), anti-NLRP3 NBP1 (1:1000, NBP1-77,080 Novus Biological), anti-ASC AL177 (1:1000, Adipogen), anti-Gasdermin D (L60) (1:1000, Cell Signaling) and anti-β-Actin (1:10,000, Abgent) were incubated over-night. β-Actin was used for normalization. Membranes were incubated with peroxidase-conjugated secondary antibody (DAKO, USA). Protein bands were visualized with the enhanced chemiluminescence (Pico or Femto, Pierce ThermoFisher Scientific, Waltham, MA USA) reagent and digitized using iBright FL1500 Imaging System (ThermoFisher, USA). Densitometric analysis was performed on background subtracted blots using Image J.
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4

Western Blot Analysis of Protein Signaling

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WB analysis was performed as previously described [18 (link)]. First, the lysed cell supernatant was run on SDS-PAGE and then blotted with antibodies. Antibodies used in WB were as follows: anti-GP73 (Abcam, Cambridge, UK), anti-P65 (Cell Signaling Technology, Boston, MA, USA), anti-phosphorylated P65 (ImmunoWay Biotechnology Company, Plano, TX, USA), anti-IκBα, anti-phosphorylated IκBα, anti-IKKα/β (Santa Cruz Biotechnology, CA, USA), anti-GAPDH, and anti-β-actin (Abgent, San Diego, CA, USA).
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5

Immunoblot Analysis of Protein Expression

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Immunoblot analysis were performed as described previously (Lee et al., 2021 (link)). Cells were harvested and lysed with RIPA buffer (Sigma) containing protease and phosphatase inhibitors (Roche). Proteins were separated by SDS-PAGE, transferred onto PVDF membranes, and blocked with 5% skim milk in TBS supplemented with 0.1% Tween-20 (TBS-Tw) for 1 h at room temperature. The membranes were then incubated with primary antibodies (Cell Signaling Technology, 1:1000 dilution) at 4°C overnight, followed by HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Cell Signaling Technology) for 1 h at room temperature. Anti-β-actin (Abgent) antibody was used as a loading control.
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6

Targeting Slug and LSD1 in TNBC

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TNBC cell lines were lentivirally transduced with the following plasmids: pLKO-SCR-sh and pLKO-Slug-sh [34] (link) and pLKO-Twist2-sh and pGIPZ-LSD1-sh-H6 (purchased from Open Biosystems, Lafayette, CO).
Slug, LSD1, and anti–β-actin expression in transduced cell lines was detected by anti-Slug (Abgent, San Diego, CA; #AP2053a), anti-LSD1 (Abcam, Cambridge, UK; #ab17721), and anti–β-actin (Santa Cruz Biotechnology, Santa Cruz, CA; #sc-47778) antibodies.
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7

Exosome Protein Expression Analysis

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Western blot analysis was carried out for the detection of CD63, CD81, TSG101, HIF-1α, COL4A1, TGFBI, GAPDH or β-actin protein levels as previously described (Yuan et al. 2010 (link), Liao et al. 2013 (link), Liu et al. 2016b , Lin et al. 2018) (link). Briefly, 30 μL exosome lysate or 30 μg of protein from each cell layer extract was loaded onto the same SDS PAGE and then transferred to a PVDF membrane. After blocking with 5% non-fat milk, the membrane was incubated with primary antibodies overnight at 4°C. The primary antibodies include anti-CD63 (1:200; Santa Cruz Biotechnology), anti-CD81 (1:200; Santa Cruz Biotechnology), anti-TSG101 (1:200; Santa Cruz Biotechnology), anti-HIF-1α (1:500; Novus, Centennial, CO, USA), anti-COL4A1 (1:1000; GeneTex, Irvine, CA, USA), anti-TGFBI (1:250; Novus), anti-GAPDH (1:5000; Abgent, San Diego, CA, USA) and anti-β-actin (1:5000; Abgent). The next morning, the membrane was washed with PBS three times every 10 min. The membrane was then incubated with the HRP-conjugated goat-anti-rabbit (1:5000; Santa Cruz Biotechnology) or HRP-conjugated goat-anti-mouse (1:5000; Santa Cruz Biotechnology) antibody in 2% non-fat milk for 1 h. Blots were processed using an ECL kit, exposed to film and then analysed by densitometry.
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