β actin mouse mab

Manufactured by Cell Signaling Technology

Sourced in United States

β-actin mouse mAb is a monoclonal antibody that specifically recognizes the β-actin protein in various species. β-actin is a widely expressed housekeeping protein and is commonly used as a loading control in Western blot analysis.

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14 protocols using β actin mouse mab

Quantifying Protein Expression Levels

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Western blot analyses were used to assess protein expression of c-Myc and cyclin B1. Three independent experiments for each cell line and condition were carried out. Total proteins were extracted using RIPA buffer (Thermo Scientific, Rockford, USA) supplemented with Halt^TM Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Rockford, IL, USA). A total of 20 μg protein was resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. After membrane blocking, the incubation with primary antibody was carried out with c-Myc (D84C12) Rabbit mAb (#5605, Cell Signaling, Danvers, USA) (dilution 1/1000), cyclin B1 Mouse mAb (sc-245, Santa Cruz Biotechnology, Santa Cruz, USA) (dilution 1/500), and β-Actin mouse mAb (#3700, Cell Signaling, Danvers, USA) (dilution 1/10,000), followed by incubation with a peroxidase-conjugated goat anti-rabbit antibody (#7074, Cell Signaling, Danvers, USA) (dilution 1/5,000) or horse anti-mouse (#7076, Cell Signaling, Danvers, USA) (dilution 1/4000 for cyclin B1 and 1/20,000 for β-Actin). Visualization of signal was carried out with an ECL plus chemoluminescence detection system (GE Healthcare, Buckinghamshire, UK). β-Actin level was used as loading control.

Sandén E., Dyberg C., Krona C., Gallo-Oller G., Olsen T.K., Enríquez Pérez J., Wickström M., Estekizadeh A., Kool M., Visse E., Ekström T.J., Siesjö P., Inge Johnsen J, & Darabi A. (2017). Establishment and characterization of an orthotopic patient-derived Group 3 medulloblastoma model for preclinical drug evaluation. Scientific Reports, 7, 46366.

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Comprehensive Protein Extraction and Analysis

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Cells were lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% NP-40) supplemented with protease inhibitor cocktail. Cell lysates were centrifuged at 12,000 rpm for 30 min at 4 °C, supernatants were saved, and protein concentrations were determined by BCA protein assay (Thermo Scientific, Rockford, Illinois, USA). Equal amounts of total cell lysates or CM of cell culture were subjected to western blot assays as we described previously [41 (link), 42 (link)] to measure protein expression. Antibodies for western blot analyses were from the following sources: NNMT rabbit mAb (E6N2Z, #33361), SIRT1 mouse mAb (1F3, #8469), Hexokinase II rabbit mAb (C64G5, #2867), LDHA rabbit mAb (C4B5, #3582), c-myc rabbit mAb (E5Q6W, #18583), BAX Rabbit mAb (D2E11, #5023), Bim Rabbit mAb (C34C5, #2933), Bcl2 Rabbit mAb (D55G8 #4223), FOXO3a rabbit mAb (D19A7, #12829), p53 Mouse mAb (1C12, #2524), Acetylated-Lysine Antibody (#9441), and β-actin mouse mAb (8H10D10, #3700) (Cell Signaling Technology, Beverly, MA, USA).

Wang J., Liu X., Huang Y., Li P., Yang M., Zeng S., Chen D., Wang Q., Liu H., Luo K, & Deng J. (2022). Targeting nicotinamide N-methyltransferase overcomes resistance to EGFR-TKI in non-small cell lung cancer cells. Cell Death Discovery, 8, 170.

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Apoptosis Induction in HL60 Cells

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HL60 cells were seeded at a 1 × 10⁶ cells/mL density onto a petri dish and incubated for 24 h. After treatments with LEO and puromycin at EC₅₀ concentrations, the cells were lysed with a RIPA buffer on ice for 30 m. Protein concentrations were determined by the Bradford test [44 (link)]. Samples were run on 10% SDS-PAGE and then electrotransferred onto a pure nitrocellulose membrane (0.45 µm). The membrane was blocked with 5% bovine serum albumin and immunoblotted with cleaved caspase-3 rabbit mAb and β-actin mouse mAb (Cell Signaling Technology, Leiden, The Netherlands). Anti-rabbit IgG and anti-mouse IgG HRP conjugate antibodies (Promega, Madison, WI, US) were used to detect the corresponding bands by acquiring images via a ChemiDoc XRS+ System (Bio Rad, Hercules, CA, Italy).

Laghezza Masci V., Ovidi E., Taddei A.R., Turchetti G., Tiezzi A., Giacomello P, & Garzoli S. (2020). Apoptotic Effects on HL60 Human Leukaemia Cells Induced by Lavandin Essential Oil Treatment. Molecules, 25(3), 538.

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Characterizing Human Microglial Cell Responses

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The HMC3 human microglial cell line (ATCC CRL-3304) and EMEM medium (ATCC 30–2003) were purchased from American Type Culture Collection (ATCC, Manassas, VA). BrdU (59–14-3) and mouse anti-BrdU (B2531) monoclonal antibody (mAb) were purchased from Sigma (St. Louise, MO). BAY 11–7082 was obtained from Cayman Chemical (Ann Arbor, MI, cat# 10010266). Human IL-6 DuoSet ELISA (DY206) kits were purchased from R&D Systems (Minneapolis, MN). Phospho-p53 (Ser 15) antibody (#9284), p53 mouse mAb (#2524), NF-κB p65 rabbit mAb (#8242), p21 Waf1/Cip1 rabbit mAb (#2947), and β-Actin mouse mAb (#3700) were purchased from Cell Signaling (Danvers, MA). Alexa Fluor-594 labeled goat anti-mouse IgG (#A10680) and Alexa Fluor-647 labeled goat anti-rabbit IgG (#A21244) were purchased from Thermo Fisher (Waltham, MA). Mouse anti-human p16 mAb (cat# 51–1325GR) was obtained from BD Biosciences. Mouse anti-human CD68 mAb (Cat# 14–0688-82, clone: KP1) was obtained from Thermo Fisher. Pro-Long Gold Anti-Fade Reagent (#9071) was obtained from Cell Signaling Technology (Danvers, MA).

Cook M., Lin H., Mishra S.K, & Wang G.Y. (2022). BAY 11-7082 inhibits the secretion of interleukin-6 by senescent human microglia. Biochemical and biophysical research communications, 617(Pt 1), 30-35.

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Western Blot Analysis of Phosphorylated Signaling Proteins

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Cell lysates containing 20-40 μg total protein were mixed with protein loading buffer, heated for 5 min at 95 °C, and subjected to SDS-polyacrylamide (12%) gel electrophoresis. Proteins were transferred onto nitrocellulose mini membrane using Trans-Blot^® Turbo™ Transfer System (Bio-Rad, CA, USA). Membranes were blocked with 5% BSA in TBST for 1 h at room temperature, incubated with specific primary antibody (concentrations according to suppliers) at 4 °C overnight, washed with TBST (3 × 15 min), incubated with horseradish peroxidase (HRP)-linked secondary antibody for 1 h at room temperature, and washed with TBST (3 × 15 min). Enhanced chemiluminescence detection was carried out using Supersignal^® West Pico Chemiluminescent Substrate Kits (1856135, 1856136, Thermo Scientific), and the blots were visualized using ChemiDoc™ MP Imaging System (Bio-Rad, CA, USA). The following primary antibodies were used: Phospho-p44/42 MAPK XP^® Rabbit mAb (#4370, Cell Signaling, Danvers, MA, USA), phosphor-Akt XP^® Rabbit mAb (#4060, Cell Signaling, Danvers, MA, USA), and β-Actin Mouse mAb (#3700, Cell Signaling, Danvers, MA, USA).

Fu J., Xia A, & Qi X. (2016). Identification of novel peptoid agonists of fibroblast growth factor receptor using microarray-based screening. MedChemComm, 7, 1183-1189.

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Antibody Characterization for Neural Studies

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Primary antibodies for immunocytochemistry included polyclonal anti-microtubule associated protein-2 (MAP-2, cat: AB5622) and monoclonal anti-glial fibrillary acidic protein (GFAP, cat: MAB360), both purchased from Millipore (Temecula, CA, USA).
Secondary antibodies used for immunocytochemistry included Alexa Fluor® 488 conjugated AffiniPure Donkey Anti-Rabbit IgG and Cy™3-conjugated AffiniPure Donkey Anti-Mouse IgG. These antibodies, along with normal donkey serum and normal goat serum, were purchased from Jackson Immuno Research (West Grove, PA, USA).
Antibodies used for ELISA were MAP-2 (monoclonal, cat: 13–1500) and Goat anti-Mouse IgG (H+L) Secondary Antibody (HRP conjugate) from Thermo Fisher Scientific (Waltham, MA, USA).
Primary antibodies for western blotting EAAT1 Rabbit mAb (1:100, cat: 5684), EAAT3 Rabbit mAb (1: 100, cat: 14501) and β-actin mouse mAb (1:1,000), were purchased from Cell Signaling (Danvers, MA, USA). EAAT2 rabbit polyclonal (1: 1,000, cat: AGC-022) was purchased from Alomone, Jerusalem, Israel. Infrared-conjugated secondary antibodies [(Anti-rabbit DyLight 800 (Green) and Anti-mouse DyLight 680 (Red), both at 1:1,000)] were used for imaging using LI-COR Image Studio, version 4.0.21 (LI-COR Biosciences; Lincoln, NE, USA).

Falcucci R.M., Wertz R., Green J.L., Meucci O., Salvino J, & Fontana A.C. (2019). Novel positive allosteric modulators of glutamate transport have neuroprotective properties in an in vitro excitotoxic model. ACS chemical neuroscience, 10(8), 3437-3453.

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Protein Expression Analysis by Western Blot

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H3K4me3 rabbit mAb, E-cadherin mouse mAb, N-cadherin rabbit mAb, and β-actin mouse mAb were purchased from Cell Signaling Technology (Danvers, MA, USA). The cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 1% Nonidet P 40 with a mixture of protease inhibitors before Western blot assay.

Sui X., Zhou H., Zhu L., Wang D., Fan S, & Zhao W. (2017). CUL4A promotes proliferation and metastasis of colorectal cancer cells by regulating H3K4 trimethylation in epithelial–mesenchymal transition. OncoTargets and therapy, 10, 735-743.

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Protein Quantification in Cells

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AKT2 rabbit mAb, PPARγ rabbit mAb, Acetyl‐CoA carboxylase rabbit mAb, and β‐actin mouse mAb were purchased from Cell Signaling Technology (Beverly, MA). Rabbit anti‐FAS and SREBP1 mouse mAb were from Abcam Inc (Cambridge, MA). Horseradish peroxidase‐conjugated, donkey anti‐Rabbit IgG and donkey anti‐Mouse IgG were purchased from Jackson ImmunoResearch (West Grove, PA).

Li S., Liu R., Pan Q., Wang G., Cheng D., Yang J., Chen H, & Xu G. (2020). De novo lipogenesis is elicited dramatically in human hepatocellular carcinoma especially in hepatitis C virus‐induced hepatocellular carcinoma. MedComm, 1(2), 178-187.

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Quantifying Inflammatory Cytokine Synthesis

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Protein synthesis of inflammatory cytokines in mixed-cultures was measured by Western blots. 35 μg per lane (IL-1β) or 5 μg per lane (CXCL10) of total protein extracted from mixed-cultures was run on Novex 10–20% Tris-Glycine gels (Life Technologies) at 120 V. Gels were transferred to Immobilon-FL membranes (Millipore) and blocked with 5% BSA for 1 h. Membranes were incubated o/n with β-actin mouse mAB 1:4000 (Cell Signaling, #3700) and either IL-1β Rabbit mAB 1:1000 (Cell Signaling, #12426) or Anti-IP10 (CXCL10) Rabbit 1:1000 (Abcam, #ab9938). After 4x washing with TBST, membranes were incubated for 1 h with DyLight 680 Anti-rabbit and 800 Anti-mouse 1:15000 ea (Cell Signaling), then visualized.

Zhu X., Levasseur P.R., Michaelis K.A., Burfeind K.G, & Marks D.L. (2016). A distinct brain pathway links viral RNA exposure to sickness behavior. Scientific Reports, 6, 29885.

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Western Blot Analysis of EMT Markers

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The cells were lysed using the mammalian protein extraction reagent, RIPA (Beyotime, Shanghai, China). Equivalent amounts (40 µg) of cell protein lysates were electrophoresed on 10% SDS-polyacrylamide gel, transferred to a PVDF membrane. Then the membrane was incubated with primary antibodies with TSPAN7 (1:1,000, Abcam, Massachusetts, USA), N-cadherin (1:2,000, CST, Massachusetts, USA), E-cadherin (1:2,000, CST), vimentin (1:2,000, CST), and tubulin (1:2,000, CST) overnight at 4°C and followed incubation by horseradish peroxidase-labeled secondary antibody. Tubulin was used as a protein-loading control. The immune complexes were tested by chemiluminescence. E-cadherin mouse mAb, N-cadherin rabbit mAb, and β-actin mouse mAb were purchased from Cell Signaling Technology. The cells were lysed in a buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EGTA, and 1% NP-40 with a mixture of protease inhibitors before Western blot assay.

Wang X., Lin M., Zhao J., Zhu S., Xu M, & Zhou X. (2018). TSPAN7 promotes the migration and proliferation of lung cancer cells via epithelial-to-mesenchymal transition. OncoTargets and therapy, 11, 8815-8822.

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