After 24 h post-transfection, cells were collected, and protein extracts obtained by adding Leammli sample buffer (Bio Rad, Ca, USA). Equal amounts of proteins were loaded and separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked using 7.5% milk in TBS/0.01% Tween buffer and then incubated with the indicated antibodies. Primary antibodies were prepared in TBS/0.01% Tween buffer as follows: anti-GAPDH (1:1000) (32233; Santa Cruz Biotechnology, Santa Cruz, California), anti-HA (1:1000) (C29F4; Cell Signaling Technology Europe, B.V), anti-p52NFκB (1:1000) (298; Santa Cruz Biotechnology, Santa Cruz, California), α-Actinin (1:1000) (17829; Santa Cruz Biotechnology, Santa Cruz, California) and Phospho-IκBα (Ser32/36)(5A5) (9246; Cell signaling Technology Europe, B.V) Membranes were washed three times with TBS/0.01% Tween buffer and incubated with HRP coupled secondary anti-mouse or anti-rabbit antibodies (Santa Cruz Biotechnology, Santa Cruz, California). Finally, Proteins were visualized using the ChemoLuminiscent Reagent (Merck Millipore, Burlington, Massachusetts, U.S) according to the manufacturer’s instructions.
α actinin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
α-actinin is a structural protein that plays a key role in the organization and dynamics of the actin cytoskeleton. It functions as a cross-linking and bundling protein, helping to maintain the integrity and structural organization of actin filaments. α-actinin is found in a variety of cell types, including muscle and non-muscle cells, where it contributes to the regulation of cell shape, motility, and adhesion.
Automatically generated - may contain errors
Lab products found in correlation
γ h2ax, by Abcam (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Polyvinylidine difluoride membrane, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Cleaved poly adp ribose polymerase, by Cell Signaling Technology (2 mentions)
Hif 1α, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Cyclin d1, by Abcam (2 mentions)
Hsp90 α β, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Srsf3, by Santa Cruz Biotechnology (2 mentions)
Twist, by Santa Cruz Biotechnology (2 mentions)
P histone h3, by Cell Signaling Technology (2 mentions)
Cyclin d1, by Santa Cruz Biotechnology (2 mentions)
Myhc iib efluor 660, by Thermo Fisher Scientific (2 mentions)
P drp1, by Cell Signaling Technology (2 mentions)
Phospho iκbα ser32 36 5a5, by Cell Signaling Technology (1 mentions)
Chemoluminiscent reagent, by Merck Group (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Acetyl coa carboxylase, by Cell Signaling Technology (1 mentions)
31 protocols using α actinin
1
Western Blot Analysis of Protein Expression
2
Protein Expression Analysis in Various Cell Lines
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HeLa, HEC-1-A, HEK293, A549, and Beas-2B cells were lysed in RIPA buffer (100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, and 1% Triton 100) at 4 °C. Proteins in the resultant lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against α-actinin (ACTN), ATF3, c-Fos, FASN HuR, Nrf2, p21, p53, p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl-CoA carboxylase (ACC), p-ACC (phosphorylation at Ser 79), ERK, p-ERK, HIF-1α, LC3B, cleaved poly-ADP-ribose polymerase (cPARP) (Cell Signaling, Danvers, MA, USA), Cyclin D1 (Abcam, Cambridge, UK), HO-1 (Enzo Life Sciences, Farmingdale, NY, USA), and DEC1 (Bethyl Laboratories, Montgomery, TX, USA).
3
Antibody panel for protein analysis
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Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).
4
Western Blot Analysis of Cellular Proteins
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The cell was lysed with RIPA (radioimmunoprecipitation assay) lysis buffer (150 mM NaCl, 0.1% SDS, 100 mM Tris-HCl of pH 8.0, and 1% Triton X-100) and then centrifuged at 12,000 r.p.m., 4°C for 15 min. The supernatant of each sample was mixed with protein loading dye, equally loaded into and subsequently separated by SDS-PAGE, transferred to a polyvinylidine difluoride membrane (Millipore, USA), and finally incubated with primary antibodies against ATF3, p21, p53, cyclin D1, α-actinin (ACTN), histone H3 phosphorylation (at serine 10), HSP90α/β (Santa Cruz Biotechnology, USA), PARP, LC3B (light chain 3B) (Cell Signaling, USA), and γH2A.x (phosphorylated form of H2A.x at serine 139) (Epitomics, USA) at 4°C overnight, followed by the secondary antibody incubation and ECL detection.
5
Cardiac Cell Phenotyping by Immunostaining
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Cells were fixed in 4% paraformaldehyde (Millipore-Sigma, St. Louis, MO) for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature for nuclear markers. Cell fixation and permeabilization for cytoskeletal markers was done with cold (−20°C) methanol for 3–5 min. Samples were washed three times (5 min each time) with PBS between each step and blocked with 3% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS for 30 min. Samples were incubated at 4°C with primary antibodies for: cardiac troponin T (TNNT2; rabbit; ab45932; Abcam, Cambridge, MA), α-actinin (ACTN1; mouse; sc-15335) and GATA4 (rabbit; sc-9053; both from Santa Cruz Biotechnology, Dallas, TX). Incubation with secondary antibodies was performed at room temperature for 1 h with donkey anti-rabbit or anti-mouse antibodies conjugated to DyLight 488 or 549 (Jackson ImmunoResearch Inc., West Grove, PA). Nuclear DNA was stained with DAPI (Sigma-Aldrich, St. Louis, MO). Controls were stained with IgG instead of with a primary antibody. Immunostaining was visualized with a Leica TCS SPE confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL).
6
Comprehensive Immunoblotting Protocol
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The primary antibodies used were as follows: SMC1A (Bethyl Laboratories, A300-055A), SMC3 (Bethyl Laboratories, A300-060A), acetylated SMC3 (kindly provided by Dr Prasad Jallepalli), RAD21 (SCC1) (Bethyl Laboratories, A300-080A), SCC-112 (PDS5) (Bethyl Laboratories, A300-089A), OBFC1 (STN1) (Novus Biologicals, NBP2-01006), CTC1 (45 (link)), α-tubulin (MilliporeSigma, T-9026), TEN1 (47 (link)), MAD2 (Bethyl Laboratories, A300-301A), α-actinin (Santa Cruz Biotechnology, SC17829), H3 (Cell Signaling Technology, 9715), Flag (Thermo Fisher Scientific, MA1-91878, PA1-984B), PolA1 (Bethyl Laboratories, A302-805A), MCM7 (Santa Cruz Biotechnology, sc22782), p53 (Cell Signaling Technology, 2524S), and p21 (Santa Cruz Biotechnology, sc-6246).
7
Immunofluorescence Analysis of Cardiac Cells
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Briefly, the cell samples were first fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 3% bovine serum albumin (BSA). After 3 rinses with PBS for 5 min, the samples were incubated with primary antibodies against cTnT (1:100; Santa Cruz Biotechnology, USA) and α-actinin (1:100; Santa Cruz Biotechnology, USA) overnight at 4˚C. After being rinsed 3 times with PBS, secondary goat anti-mouse IgG Alexa Fluor594 and goat anti-rabbit IgG Alexa Fluor488 (1:1000; Invitrogen, USA) antibodies were applied for 1 hour at room temperature in the dark. Nuclei of all samples were stained with 4,6-diamidino-2-phenylindole (DAPI; Abcam, USA) for 5 min. Fluorescence images were captured with Leica TCS SP5 MP confocal laser scanning microscope (Leica, Wetzlar, Germany).
8
Immunofluorescent Cardiac Cell Staining
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After treatment, cardiac cells were fixed with 4% paraformaldehyde for 20 min and stained with α-actinin (sc-17829, Santa Cruz Biotechnology, diluted 1:50) at 4 °C overnight. After washed with PBS, the cells were incubated with FITC-conjugated goat anti-mouse antibody (A0568, Beyotime, diluted 1:300) in the dark. Heart tissues were stained with WGA Alexa Fluor™ 555 Conjugate (500 μg/ml, W32464, Thermo Scientific) at room temperature for 10 min and washed with PBS four times. Cells and tissue sections were imaged using a confocal fluorescence microscope (Leica).
9
Characterization of Lung Cancer Cell Lines
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Normal lung epithelial cell line NL20 and NSCLC cell lines were previously described.44 (link) NCI-H1299 cells (American Type Culture Collection, ATCC) were grown in DMEM (Sigma) supplemented with 10% FBS (PAA); A549 cells (kindly provided by Dr. Susan Cole, Queen’s University) were grown in RPMI (Sigma) supplemented with 5% FBS and 1% L-glutamine. CIP4 antibodies include rabbit anti-CIP4 Ab#1 used for immunoblotting,11 (link) and rabbit anti-CIP4 Ab#2 used for immunohistochemistry (raised and affinity purified using the peptide QDTPIYTEFDEDFEE, Open Biosystems). Commercial antibodies included: CIP4, N-WASP, pS2448-mTOR, pS473-Akt, pT308-Akt and Akt1/2 were from Cell Signaling Technology; EGFR, pY1068-EGFR and PE-conjugated mouse anti-human EGF receptor were from BD Bioscience; ERK1, pERK, β-actin, MMP-2, α-actinin, and EGFR were from Santa Cruz Biotechnology.
10
Western Blot Antibody Protocol
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Antibodies used for western blotting were purchased from Cell Signaling (Danvers, MA, USA).
RNA mimics were obtained from Dharmacon (Lafayette, CO). All other reagents were of molecular biology grade. Catalog numbers for siRNA, antibodies, and other reagents used in this study can be found in the supplement materials (Supplement TableS2 ). These antibodies include the following: (G6PD, Abcam, cat# ab76598), (α-actinin, Santa Cruz, Sc-17829), (Vinculin, Protein-Tech, 66305-1-Ig), (Gapdh, Cell Signaling, 5174 L).
RNA mimics were obtained from Dharmacon (Lafayette, CO). All other reagents were of molecular biology grade. Catalog numbers for siRNA, antibodies, and other reagents used in this study can be found in the supplement materials (Supplement Table
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