α actinin

Manufactured by Thermo Fisher Scientific

Sourced in United States

α-actinin is a structural protein that is involved in the crosslinking and organization of actin filaments within the cytoskeleton. It plays a role in the assembly and maintenance of the contractile apparatus in muscle cells and the organization of the actin cytoskeleton in non-muscle cells.

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8 protocols using α actinin

Protein Expression Analysis in Cardiac Tissue

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Ventricular tissue was homogenized in RIPA buffer (10 mmol/L Tris-Cl (pH
8.0), 1mmol/L EDTA, 0.5 EGTA, 1% Triton X-100, 0.1% sodium
deoxycholate, 0.1% SDS, 140mmol/L NaCl) that was supplemented with
various inhibitors: sodium vanadate, leupeptin, aprotinin,
p-nitrophenyl phosphate, and phenylmethylsulfonyl fluoride.
Western blot analysis was performed according to protocols described previously
[9 (link)]. The antibodies
for immunoblotting were as follows: CaMKIIδ (D.M. Bers, UC Davis); GAPDH
(CST); phospho-IKKα/β at its autophosphorylation site
(Ser176/180) (CST); phospho-CaMKII at its autophosphorylation site (Thr286)
(Thermo); NF-κB p65 (CST); α-actinin (CST); RhoGDI (CST); Lamin
A/C (CST); IκBα (CST)

Gray C.B., Suetomi T., Xiang S., Mishra S., Blackwood E.A., Glembotski C.C., Miyamoto S., Westenbrink B.D, & Brown J.H. (2017). CaMKIIδ subtypes differentially regulate infarct formation following ex vivo myocardial ischemia/reperfusion through NF-κB and TNF-α. Journal of molecular and cellular cardiology, 103, 48-55.

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Characterization of Myogenic Markers

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The following antibodies were used for immunofluorescence or western blot: pan-MyHC (MF20; DSHB), Titin (9D10; DSHB), MyHC-neo (N3.36; DSHB) (MHCN; Leica), MYH3 (F1.652; DSHB), MYH1/2 (SC-71; DSHB), α-actinin (MA122863; Thermofisher), Desmin (sc-23879; SCBT), ACTB (sc-4778; SCBT), OCT3/4 (C-10; SCBT), SOX2 (Y-17; SCBT), NANOG (H-2; SCBT), Alexa Fluor 488 Phalloidin (A12379; Thermofisher), Alexa fluor 555 goat anti-mouse IgG (A-21424; Thermo Fisher), Alexa fluor 488 goat anti-rabbit IgG (A-11008; Thermo Fisher), and mouse IgG HRP-linked (NA931; GE Healthcare).

Selvaraj S., Mondragon-Gonzalez R., Xu B., Magli A., Kim H., Lainé J., Kiley J., Mckee H., Rinaldi F., Aho J., Tabti N., Shen W, & Perlingeiro R.C. (2019). Screening identifies small molecules that enhance the maturation of human pluripotent stem cell-derived myotubes. eLife, 8, e47970.

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Immunohistochemical Characterization of Engineered Smooth Muscle Tissue

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Collagen IV was immunostained with primary antibodies against collagen IV (Sigma-Aldrich, St. Louis, MO, USA) and α-actinin (Thermo Fisher Scientific, Waltham, MA, USA) after fixing the trained eSMTs with 4% paraformaldehyde (Santa Cruz Biotechnology, Dallas, TX, USA), and permeabilizing them with 0.2% Triton-X (Thermo Fisher Scientific, Waltham, MA, USA). Trained eSMTs were incubated with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) to block the nonspecific binding of antibodies. Hoechst (Thermo Fisher Scientific, Waltham, MA, USA) and rhodamine phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were applied together for 30 minutes to visualize nucleus and actin, respectively.

Kim H., Kim M.C, & Asada H.H. (2019). Extracellular matrix remodelling induced by alternating electrical and mechanical stimulations increases the contraction of engineered skeletal muscle tissues. Scientific Reports, 9, 2732.

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3D Reconstruction of Cardiac Embryos

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Embryos were fixed with 4% of paraformaldehyde overnight, dehydrated and embedded in paraffin, and cut at 8 µm slice thickness. One in four sections were used for immunohistochemistry to identify the myocardium by an overnight incubation at room temperature with mouse‐α‐actinin (Sigma‐Aldrich A9357, St. Louis, MO, US, diluted 1:400), followed by a 2‐hour incubation at room temperature with a goat‐anti‐mouse antibody conjugated with Alexa 488 (Thermo Fisher, Waltham, MA, US, diluted 1:250). DAPI was used for staining nuclei (Thermo Fisher, Waltham, MA, US, diluted 1:1000). Labelling α‐actinin‐positive cardiac tissue, calculating volume and generating 3D reconstructions were performed using Amira Software (version 5.3.3, Thermo Fisher Scientific, Waltham, MA, US) as described before.¹⁸ Cardiac nuclei count was determined using previously described custom software.⁵⁷

Marchal G.A., Verkerk A.O., Mohan R.A., Wolswinkel R., Boukens B.J, & Remme C.A. (2020). The sodium channel NaV1.5 impacts on early murine embryonic cardiac development, structure and function in a non‐electrogenic manner. Acta Physiologica (Oxford, England), 230(2), e13493.

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Immunofluorescent Staining of Cardiomyocytes

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For immunofluorescent staining, cells were fixed with PBS containing 4% paraformaldehyde for 20 minutes at room temperature. After washing with PBS, cells were blocked for 30 minutes with PBS containing 5% bovine serum. Staining with primary antibodies: cardiac troponin I (cTnI) (RRID: AB_2532494, ThermoFisher, Waltham, MA, USA), α-Actinin (RRID: AB_2692251, ThermoFisher, Waltham, MA, USA) diluted in blocking buffer was performed for overnight at −4 °C temperature. Secondary antibodies were used in the next day, following staining with DAPI to detect the cell nucleus. Fluorescent images were acquired using a Laser confocal microscope (Leica TCS SP8).

Lu Y., Fang Q., Qi M., Li X., Zhang X., Lin Y., Xiang Y., Fu Q, & Wang B. (2023). Copy number variation-associated lncRNAs may contribute to the etiologies of congenital heart disease. Communications Biology, 6, 189.

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Cardiomyocyte Immunofluorescence Imaging

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The cells were fixed by 4% formaldehyde followed by 5 min of permeabilization with 0.2% Triton X-100, blocked with 5% BSA for 60 min and stained with α-actinin (Invitrogen, Carlsbad, CA, USA), followed by staining with a fluorescent secondary antibody. Then, DAPI was used to label the nuclei. Immunofluorescence images were acquired by Olympus laser confocal microscope (FV 1000, Olympus, Tokyo, Japan). Cardiomyocytes surface area was determined from more than 100 cells each group using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) by total cell area divided total cell number, as previously described [17 (link)].

Yang Y., Wang X., Yan P., Wang D., Luo T., Zhou Y., Chen S., Liu Q., Hou J, & Wang P. (2023). Transmembrane protein 117 knockdown protects against angiotensin-II-induced cardiac hypertrophy. Hypertension Research, 46(10), 2326-2339.

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Cardiomyocyte Morphometry Imaging Protocol

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The cells were fixed by 4% formaldehyde followed by 5 min of permeabilization with 0.2% Triton X-100. Then, cells were stained with α-actinin (Invitrogen, Carlsbad, CA, USA), followed by staining with a fluorescent secondary antibody. Immunofluorescence images were acquired by Olympus laser confocal microscope (FV 1000, Olympus, Tokyo, Japan). Cardiomyocytes surface area was determined from more than 100 cells each group using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) by total cell area divided total cell number.

Yang Y., Du J., Xu R., Shen Y., Yang D., Li D., Hu H., Pei H, & Yang Y. (2020). Melatonin alleviates angiotensin-II-induced cardiac hypertrophy via activating MICU1 pathway. Aging (Albany NY), 13(1), 493-515.

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Immunofluorescent Staining of Cardiac Cells

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Cells were first fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 15 min at room temperature (RT), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich: Merck KGaA) for 10 min at RT and blocked with 10% goat serum (Sigma-Aldrich; Merck KGaA) at RT for 1 h. Samples were subsequently stained with primary antibodies against cardiac troponin T (cTnT; 1:200; cat. no. 710635; Invitrogen; Thermo Fisher Scientific, Inc.) and α-actinin (Invitrogen; 1:200; cat. no. 710947; Thermo Fisher Scientific, Inc.), dissolved in PBS at 4°C for 12 h and further incubated with Alexa Fluor™ 555-conjugated antibody (1:500; cat. no. A0460; Beyotime Institute of Biotechnology) and Alexa Fluor™ 488-conjugated antibody (1:500; cat. no. A0423; Beyotime Institute of Biotechnology) at RT for 2 h. Nuclei were stained using 1 µg/ml DAPI (Sigma-Aldrich; Merck KGaA) at RT for 5 min. An Optiphot-2 microscope (Nikon Corporation) equipped with a CCD video camera system (Optronics Engineering, Ltd.) and a computer interface (NIS-Elements; version 4.2.0; Nikon Corporation) was used for imaging analysis (magnification, ×100). In total, 40 images were taken for each well.

Ke M., Ji M., Wang H., Yao Y., Wu Y, & Qi N. (2020). Inhibition of Rho-associated protein kinase improves the survival of human induced pluripotent stem cell-derived cardiomyocytes after dissociation. Experimental and Therapeutic Medicine, 19(3), 1701-1710.

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