Total EPCs protein were extracted and quantified by protein extraction reagent (Merck) and bicinchoninic acid protein assay kit (Thermo Fisher) separately. Protein extracts were subjected to SDS‐PAGE, transferred to polyvinylidene fluoride membranes (Roche). The following antibodies were used: rabbit anti‐CXCR4 antibody (1:500; ABCAM, USA), rabbit anti‐actin antibody (1:2000; Cell Signaling Technology), rabbit anti‐VEGFa antibody rabbit (1:500; Santa Cruz, USA), rabbit anti pan protein kinase B (1:1000, Abcam), p‐Akt (1:2000, Ser473; Abcam), rabbit anti human PDGF B (1:2000, Abcam), goat anti human FGF 23 (1:800, Abcam), mouse anti human eNOS (1:500 Abcam), p‐eNOS (1:500 Ser1177; Abcam), and rabbit anti‐GADPH antibody (1:3000; Cell Signaling Technology). Proteins were visualized with HRP‐conjugated anti‐rabbit or anti goat IgG (1:2000; Cell Signaling Technology), followed by use of the ECL chemiluminescence system (Thermo).
Rabbit anti actin antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
The Rabbit anti-actin antibody is a primary antibody that recognizes actin, a highly conserved cytoskeletal protein found in all eukaryotic cells. This antibody is useful for the detection and visualization of actin in various experimental applications, such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Roche (4 mentions)
Hrp conjugated anti rabbit igg, by Cell Signaling Technology (3 mentions)
Rabbit anti gadph antibody, by Cell Signaling Technology (2 mentions)
Ecl chemiluminescence system, by Thermo Fisher Scientific (2 mentions)
Rabbit anti cxcr4 antibody, by Abcam (2 mentions)
Alexa fluor 680 goat anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Mab5310, by Merck Group (2 mentions)
Irdye 800cw goat anti rabbit igg h l, by LI COR (2 mentions)
Ecl chemiluminescence system, by Cell Signaling Technology (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Rabbit anti ampk antibodies, by Abcam (2 mentions)
Rabbit anti phospho ampk antibody, by Abcam (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Irdye 680, by LI COR (2 mentions)
Irdye 800, by LI COR (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Protein extraction reagent, by Merck Group (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
P akt, by Abcam (1 mentions)
16 protocols using rabbit anti actin antibody
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of LAT1, CD98, and β-Actin
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Cells were dissolved in sample buffer (25% glycerin, 1% SDS, 62.5 mM Tris-Cl, 10 mM dithiothreitol) and incubated at 65 °C (LAT1) or 95 °C (CD98 and β-actin) for 15 min. Aliquots of samples containing 40 μg of protein were analysed by 10% SDS–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. Blots were incubated at 4 °C overnight in 10 mM Tris–HCl, 100 mM NaCl, 0.1% Tween 20, pH 7.5 (TBST), with 5% skim milk and then with rabbit anti-LAT1 C-terminus antibody (1 : 5000 dilution; Morimoto et al, 2008 (link)), rabbit anti-LAT1 N-terminus antibody (1 : 5000 dilution; Morimoto et al, 2008 (link)), rabbit anti-CD98 antibody (Santa Cruz Biotechnology, 1 : 200 dilution) or rabbit anti-actin antibody (Cell Signaling Technology, 1 : 1000 dilution) at 4 °C overnight. After having been washed with TBST, the blots were incubated with goat horseradish peroxidase-conjugated anti-rabbit IgG antibody (Cell Signaling Technology,1 : 20 000 dilution) for 1.5 h at room temperature. The blots were further washed with TBST, and specific proteins were visualised by using enhanced chemiluminescence western blotting detection reagents (GE Healthcare).
3
Rabbit Anti-CD44v3 Antibody Evaluation
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Rabbit anti-CD44v3 antibody was obtained from EMD Chemicals (Gibbstown, NJ). Other immunoreagents such as rabbit anti-cIAP1 antibody were from Abcam (Cambridge, MA) and rabbit anti-actin antibody was purchased from Cell Signaling (Beverly, MA), respectively. Cisplatin was obtained from Millipore (Darmstadt, Germany). Different sizes of research grade HA fragments (e.g., 5 kD, 20 kDa, 200 kDa, and 700 kDa) were purchased from Lifecore Biomedical (Chaska, MN).
4
Quantifying CXCR4, SIRT5, and JAK2 Proteins
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Total EPC protein were extracted and quantified by cytoBuster TM protein extraction reagent (Beyotime Biotechnology, China) and bicinchoninic acid protein assay kit (I Thermo, USA) separately. Protein extracts were subjected to SDS-PAGE, transferred to polyvinylidene fluoride membranes (Roche, Indianapolis, IN, USA). The following antibodies were used: rabbit anti-CXCR4 antibody (1:500; ABCAM, USA), rabbit anti-actin antibody (1:2000; Cell Signaling Technology), rabbit anti-SIRT5 antibody rabbit (1:500; Santa Cruz, USA), rabbit anti-Phospho-JAK2 antibody rabbit (1:1000; Immunoway, USA) and rabbit anti-GADPH antibody (1:3000; Cell Signaling Technology). Proteins were visualized with HRP-conjugated anti-rabbit IgG (1:2000; Cell Signaling Technology), followed by use of the ECL chemiluminescence system (Thermo).
5
Western Blot Analysis of Cav1.3 Protein
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Samples were collected and prepared as described previously (Ko M. L. et al., 2009 (link); Huang et al., 2013 (link); Lin et al., 2015 (link)). Retinas were homogenized in a Tris lysis solution (50 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% NP-40) including phosphatase (50 mM NaF, 1 mM Na3VO4) and protease inhibitors (Sigma-Aldrich). After centrifugation to remove cellular debris, an equal volume of 2x Laemmli buffer was added to each sample lysate, then the samples were heated at 95°C for 5 min. Proteins were separated by SDS-PAGE (10% gels) for 1–2 h. Proteins were then transferred to nitrocellulose membranes and probed by primary antibodies. The primary antibodies used were rabbit anti-Cav1.3 antibody (1:1,000; Chemicon/ Millipore Sigma) and rabbit anti-actin antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA). Actin was used for loading controls. The secondary antibody (goat anti-rabbit) conjugated to horseradish peroxidase (1:1,000; Cell Signaling Technology) and the Femto and Pico electrochemiluminescense (ECL) kits (Pierce ThermoFisher Scientific, Waltham, MA, USA) were used to visualize the blots.
6
Generation of Anti-CoA Antibody 1F10
7
Reconstitution of Influenza Ribonucleoprotein
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pcDNA plasmids for the in vitro reconstitution of the ribonucleoprotein system of influenza A/Beijing/2009 (H1N1) and A/Kurume/2009 (H1N1) were designed based on the RNA sequences of the respective viruses. pcDNA plasmids for the reconstitution of the ribonucleoprotein system of A/WSN/33(H1N1) were kindly provided by Prof. Ervin Fodor (University of Oxford, UK). Rabbit anti-influenza HA antibody was purchased from Sino Biological (Beijing, China). The mouse anti-influenza NP antibody (MAB8251) was purchased from Merck Millipore (Burlington, MA, USA). Rabbit anti-actin antibody was purchased from Cell Signalling Technology (Danvers, MA, USA). Rabbit anti-PB1, anti-PA, and anti-PB2 antibodies were purchased from GeneTex Technology (Irvine, CA, USA).
8
Probing Akt Signaling in Arbovirus-Infected Cells
9
Western Blot Analysis of KIF11 Protein
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Cells were lysed in Pierce RIPA buffer (Thermo Scientific) that included a 1% protease inhibitor cocktail (Thermo Scientific). After homogenization, the cell lysates were incubated on ice for 30 min and centrifuged at 15,000 rpm for 15 min to separate the supernatant from cellular debris. The amount of total protein was estimated using the Qubit Protein assay kit (Thermo Scientific), and the proteins were then mixed with SDS sample buffer and incubated at room temperature for 5 min after boiling at 100°C for 5 min. After electrophoresis on 10% Mini-Protean®TGX gels (Bio Rad, Hercules, CA, USA), the proteins were transferred onto Trans-Blot®Turbo 0.2 μm PVDF membranes (Bio-Rad). The membranes were blocked using the iBind solution kit (Thermo Scientific) and incubated with rabbit anti-KIF11 antibody (catalog no. HPA010568; Sigma-Aldrich, St. Louis, MO, USA) or rabbit anti-actin antibody (catalog no. 4970, 13E5; Cell Signaling Technology, Danvers, MA, USA). Membranes were incubated with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (GE Healthcare, Buckinghamshire, UK) for 60 min at room temperature. Protein bands were visualized using Image Quant LAS 4000 mini (GE Healthcare).
10
Generation and Characterization of Anti-CoA Antibody
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The generation of the anti-CoA antibody (1F10) was described recently [24 (link)]. All common chemicals were obtained from Sigma–Aldrich unless otherwise stated. The following antibodies and dilutions were used: mouse anti-CoA antibody (0.17 µg/ml); rabbit anti-actin antibody (Cell Signaling Technology #4968, 1:2000); rabbit anti-tubulin antibody (Cell Signaling Technology #2148, 1:2000); rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Abcam #ab181602, 1:6000); mouse anti-GSH antibody (Millipore #MAB5310, 1:1000) and rabbit anti-pyruvate dehydrogenase kinase 2 (PDK2) antibody (Abcam #ab68164, 0.5 µg/ml). Primary antibodies were diluted in Odyssey blocking buffer containing 0.01% Tween 20. Secondary antibodies [Alexa Fluor 680 goat anti-mouse IgG H&L (Life Technologies) and IRdye 800 CW goat anti-rabbit IgG H&L (LI-COR Biosciences)] were diluted in Odyssey blocking buffer (1:10 000) containing 0.02% sodium dodecyl sulphate (SDS).
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