Cells were washed twice in PBS, lysed in 1× Glasgow lysis buffer (GLB; 1% [vol/vol] Triton X-100, 120 mM KCl, 30 mM NaCl, 5 mM MgCl2, 10% [vol/vol] glycerol, and 10 mM PIPES-NaOH, [pH 7.2], with protease and phosphatase inhibitors) and harvested by centrifugation (2,800 × g, 10 min, and 4°C) before determination and normalization of protein concentration by bicinchoninic acid (BCA) assay (Pierce). Following separation by SDS-PAGE, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked in 50% (vol/vol) Odyssey blocking (OB) buffer (LI-COR) in Tris-buffered saline (TBS). The membrane was incubated with primary antibodies overnight at 4°C, followed by secondary antibodies for 2 h at room temperature, both prepared in 25% OB buffer. Primary antibodies used were anti-NS5A (sheep, prepared in-house) at 1:4,000 (52 (link)), anti-NAP1L1 (rabbit; Santa Cruz) at 1:350, anti-Bin1 (rabbit; Generon) at 1:300, anti-VAP-A (rabbit; Generon) at 1:1,000, anti-VAP-B (rabbit; Generon) at 1:1,000, and anti-α-actin (mouse; Sigma) at 1:10,000. Secondary antibodies were anti-rabbit, anti-sheep (800 nm), or anti-mouse (700 nm) antibodies, used at 1:10,000 prior to imaging using a LI-COR Odyssey Sa infrared imaging system. Quantification of Western blots was carried out using Image Studio v3.1 (LI-COR) using a background subtraction method.
Anti α actin
Manufactured by Merck Group
Sourced in United States, United Kingdom
Anti-α-actin is a laboratory reagent used for the detection and quantification of α-actin, a key structural protein found in eukaryotic cells. It provides a tool for researchers to investigate the role of α-actin in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscopy, by Leica camera (2 mentions)
Anti lamin a c, by Cell Signaling Technology (2 mentions)
Anti iκbα, by Santa Cruz Biotechnology (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Anti p65, by Santa Cruz Biotechnology (2 mentions)
Multi gauge software, by GE Healthcare (2 mentions)
A5106 200ul, by Thermo Fisher Scientific (2 mentions)
Anti phospho myosin light chain ser 19, by Santa Cruz Biotechnology (2 mentions)
Ac lysine antibody, by Santa Cruz Biotechnology (2 mentions)
Anti n wasp, by Santa Cruz Biotechnology (2 mentions)
Am4300, by Merck Group (2 mentions)
Anti vimentin, by BD (2 mentions)
Anti calponin, by Merck Group (2 mentions)
Anti ram 11, by Agilent Technologies (2 mentions)
Enhanced chemiluminescent reagent, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (2 mentions)
G box f3 imaging system, by Syngene (2 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (2 mentions)
Anti phospho erk, by Cell Signaling Technology (2 mentions)
Image studio v3, by LI COR (1 mentions)
34 protocols using anti α actin
1
Quantitative Western Blot Analysis
2
Immunofluorescence Staining of Adherent Cells
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Adherent cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 and then blocked with 1% BSA for 1 hr. After incubation with anti-α-actin (Sigma) as primary antibody overnight at 4°C, FITC-conjugated goat anti rabbit IgG (Abcam) was used as secondary antibody. 6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. The slides were photographed using fluorescence microscopy (Leica, Germany). The dilution concentration of the primary antibodies was 1:10 to 1:100, and the secondary antibodies at a dilution of 1:200.
3
Isoproterenol and Trichostatin A Modulate Cell Signaling
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Isoproterenol and Trichostatin A (TSA) were purchased from Sigma Aldrich (St. Louis, MO, USA) and used at 10 µM and 100 nM, respectively. Murine TNF-α was a gift from the VIB Department for Molecular Biomedical Research of Ghent University (VIB-UGent, Gent, Belgium) and was used at 2000 IU/ml. Insulin-like growth factor-1 (IGF-1) was from ImmunoTools (Friesoythe, Germany) and was used at 10 ng/ml. Anti-β2-AR, anti-TNF-R1, anti-myogenin, anti-PARP, anti-P-H3-Ser10, anti-CBP, anti-RNA polymerase II, anti-p65, anti-IκBα, anti-P-CREB-Ser133 and anti-PKAc were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-P-p65-Ser536, anti-P-ERK-Thr202/Tyr204, anti-P-JNK-Thr183/Tyr185, anti-P-p38-Thr180/Tyr182, anti-P-MSK-1-Thr581, anti-Lamin A/C and anti-CREB were from Cell Signaling Technology (Danvers, MA, USA). Anti-Ac-H3-Lys27 and GAPDH were from AbCam (Cambridge, UK). Anti-α-tubulin and anti-α-actin were from Sigma-Aldrich. In figures, the expression “antibody anti-” was substituted by the Greek letter “α-”. AatII and HincII restriction enzymes were obtained from New England BioLabs (Ipswich, MA, USA).
4
Immunoblotting Assay for Endothelial Signaling
5
Quantifying COX-2 Protein Expression
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Frozen samples of MRAs were sonicated with ice-cold RIPA buffer (Sigma Aldrich, St Louis, MO, United States). The lysate was centrifuged at 6,000 rpm, the supernatant of soluble proteins was collected, and the protein concentration was determined by Lowry assay. Laemmli solution was added to aliquots containing 80 μg of protein from each animal. The proteins were separated on a 10% SDS-polyacrylamide gel and blotted to PVDF membrane (Amersham, GE Healthcare, Buckinghamshire, United Kingdom). Blots were incubated overnight at 4°C with mouse monoclonal antibodies for COX-2 (1:200; Cayman Chemical, Ann Arbor, MI, United States). Membranes were washed and incubated with a horseradish peroxidase-coupled anti-mouse (1:5,000; Stress Gen Bioreagent Corp., Victoria, BC, Canada) antibody for 1 h at room temperature. After thoroughly washing, the bands were detected using an ECL plus Western blotting detection system (GE Healthcare) after exposure to X-ray AX film (Hyperfilm ECL International). Blots were quantified using the Image J densitometry analysis software (National Institutes of Health). Anti α-actin (1:5,000, Sigma Chemical Co.) expression was used as a loading control.
6
Western Blot Analysis of YY1 Protein
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Total cellular proteins were size-fractionated on Bis-acrylamide 12% gels (Sigma-Aldrich Saint Louis, Missouri, USA) using Mini-Cell (Biorad) and transferred onto 0.45-μm nitrocellulose membranes using Immobilon-P (Millipore, Billerica, Massachusetts, USA) according to the manufacturer’s recommendations. For this experiment we used YY1 (C20, Santa Cruz Biotechnology, Santa Cruz, USA) as primary antibody. Signal quantification was performed using Amersham Hyperfilm ECL (GE Healthcare, Little Chalfont, UK) and normalized to loading control (anti-α actin, Sigma). A dilution of 1:5000 of anti-rabbit IgG (Amersham, GE Healthcare) was used as secondary antibody.
7
Western Blot Quantification Protocol
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Western blot analysis was performed on 30 µg of total protein extracts according to the protocol previously described. Proteins were resolved on 10% SDS-page gels and transferred to nitrocellulose membranes by the Trans-blot Turbo instrument (Bio-Rad, Hercules, CA, USA). After blotting, the filters were blocked and then hybridized at 4 °C for 2 h with anti-FLAG antibody (1:5000 dilution; Sigma-Aldrich, Saint Louis, MO, USA). The membranes were washed thrice with 1× TBS-Tween 20 buffer for 5 min and incubated for 45 min with a peroxidase-conjugated anti-mouse IgG secondary antibody (1:10,000 dilution, Bio-Rad Laboratories, Hercules, CA, USA). The antigen–antibody complexes were then detected using the ECL Immobilon Western Chemiluminescent HRP-substrate system (Millipore, Darmstadt, Germany) and autoradiography, according to the manufacturer’s instructions. Signals were subsequently normalized with an antibody anti α-actin (dilution 1:10,000; Sigma-Aldrich, Saint Louis, MO, USA). Western blots bands were quantified using ImageJ software 1.53a (Bio-Rad Laboratories, Hercules, CA, USA).
8
Western Blotting and Co-Immunoprecipitation Protocols
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Western blotting of cell lysis and co‐immunoprecipitation were performed using the experimental procedures as previously described.8 , 26 , 30 , 32 , 33 Antibodies used were anti‐Abi1 (1:1500, Sigma #A5106‐200UL, L/N 076M4842V), anti‐GAPDH (1:1500, Ambion #AM4300, L/N 1311029), and anti‐α‐actin (Sigma, A2647, #22190320, 1:3000), anti‐phospho‐myosin light chain (Ser‐19, Santa Cruz Biotechnology, 1:500), Ac‐lysine Antibody (Santa Cruz Biotechnology, S7F8, sc‐81623), anti‐N‐WASP (Santa Cruz Biotechnology, #sc‐10121, L/N G2211 1:500), anti‐myosin light chain (1:1000, a gift of Dr Gunst), anti‐phospho‐N‐WASP (Y256) (EMD Millipore, #AB1966, L/N 2795491,2838736, 1:1000), and anti‐vimentin (BD Biosciences, #550515, L/N 3214517, 1:10 000). Phospho‐vimentin (Ser56) antibody (1:500) was produced as previously described.29 , 34 The antibodies were validated by examining the molecular weight of target proteins. In addition, anti‐Abi1 was validated using Abi1 KD cells.17 Finally, vendors have provided datasheet to show that antibodies were validated by positive controls. The levels of proteins were quantified by scanning densitometry of immunoblots (Fuji Multi Gauge Software or GE IQTL software). The luminescent signals from all immunoblots were within the linear range.
9
IFIT5 Silencing and VHSV Infection in Rainbow Trout RBCs
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Three different IFIT5 small interfering RNA sequences (siIFIT5) were designed and produced by Sigma-Aldrich (Table 1 ). RBCs were transfected with a mixture of 3 siIFIT5 sequences by electroporation. For each electroporation reaction, we used 187 pmol of each siRNA per 0.5*106 cells resuspended in Buffer T (Neon™ Transfection System Kit, Life Technologies). As a negative control, 187 pmol siGFP was used (Sigma-Aldrich). RBCs were electroporated as described above and incubated for 3 days at 14°C.
IFT5 silencing was evaluated at transcript and protein level. ifit5 gene silencing was analyzed by RT-PCR, described in section RNA Isolation and Gene Expression by RT-PCR and RT-qPCR. Moreover, IFIT5 silencing was determined by western blot, described in section Rainbow Trout Anti-IFIT5 Antibody Production, using anti-IFIT5 antibody at 1/500 dilution and anti-α-actin antibody (Sigma-Aldrich) (1/100) was used as endogenous control. Goat anti-mouse peroxidase and goat anti-rabbit peroxidase (Sigma-Aldrich), were respectively used for anti-IFIT5 and anti-α-actin antibodies.
Transfected RBCs were separately exposed to VHSV MOI 1 at 14°C. After 3 h of VHSV exposure, RBCs were washed with culture medium and incubated for 24 h. Cells were resuspended and stored in appropriate buffer for RNA extraction.
IFT5 silencing was evaluated at transcript and protein level. ifit5 gene silencing was analyzed by RT-PCR, described in section RNA Isolation and Gene Expression by RT-PCR and RT-qPCR. Moreover, IFIT5 silencing was determined by western blot, described in section Rainbow Trout Anti-IFIT5 Antibody Production, using anti-IFIT5 antibody at 1/500 dilution and anti-α-actin antibody (Sigma-Aldrich) (1/100) was used as endogenous control. Goat anti-mouse peroxidase and goat anti-rabbit peroxidase (Sigma-Aldrich), were respectively used for anti-IFIT5 and anti-α-actin antibodies.
Transfected RBCs were separately exposed to VHSV MOI 1 at 14°C. After 3 h of VHSV exposure, RBCs were washed with culture medium and incubated for 24 h. Cells were resuspended and stored in appropriate buffer for RNA extraction.
10
Immunofluorescence Characterization of Smooth Muscle Cells
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