The α- and β-synucleins were identified by mouse monoclonal antibody purchased from Santa Cruz, Inc. (Dallas, USA): α/β-synuclein (F-11, sc-514908, 1:500 dilution). glyceraldehyde-3P-dehydrogenase (GAPDH)- and actin-oriented antibodies were also from Santa Cruz, Inc. (Dallas, USA): GAPDH (FL-335, sc-25778, 1:5000 dilution), actin (I-19, sc-1616, 1:2500 dilution). Beta-actin polyclonal antibody was purchased from Bioss Antibodies: β-actin (bs-0061R, 1:5000 dilution). Sheep anti-mouse IgG-HRP (NA931V, 1:10000 dilution) was from GE Healthcare UK and goat anti-rabbit IgG-HRP (sc-2004, 1:10000 dilution) was from Santa Cruz, Inc. (Dallas, USA).
Beta actin
Manufactured by Bioss Antibodies
Sourced in China, United States
Beta-actin is a highly conserved cytoskeletal protein that is ubiquitously expressed in all eukaryotic cells. It is a component of the microfilament system and plays a crucial role in cell structure and motility.
Automatically generated - may contain errors
Lab products found in correlation
Nf κb, by Proteintech (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Goat anti rabbit igg hrp sc 2004, by Santa Cruz Biotechnology (1 mentions)
Gapdh fl 335 sc 25778, by Santa Cruz Biotechnology (1 mentions)
β actin bs 0061r, by Bioss Antibodies (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
P erk, by Proteintech (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Bioss Antibodies (1 mentions)
Cox 4, by Abbkine (1 mentions)
C fos, by Abcam (1 mentions)
Nfatc1, by Cell Signaling Technology (1 mentions)
Ripa solution, by Beyotime (1 mentions)
Bca protein concentration determination kit, by Solarbio (1 mentions)
Ripa lysate, by Solarbio (1 mentions)
Ribofect cp transfection reagent, by RiboBio (1 mentions)
P nf κb, by Abcam (1 mentions)
Interleukin 6 (il 6), by Proteintech (1 mentions)
Il 1β, by Proteintech (1 mentions)
10 protocols using beta actin
1
Immunoblotting of Synuclein Proteins
2
Western Blot Analysis of Protein Markers
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OCs were lysed in RIPA solution (Beyotime) to extract total proteins, which were quantified as per instructions from a BCA kit (Thermo Fisher Scientific). Protein samples (20 μg) were loaded onto 8% to 15% SDS-PAGE gel, separated, and transferred to PVDF membrane (Millipore, Shanghai). After blocking with 5% skim milk, primary antibodies against Abca1 (1:1,000, affinity, Liyang, China), Bax (1:1,000, affinity), Bcl-2 (1:1,000, affinity), beta-actin (1:3,000, Bioss, Beijing, China), Caspase-3 (1:1,000, Cell Signaling Technology), c-Fos (1:1,000, Abcam, Cambridge, UK), COX IV (1:1,000, Abbkine, Wuhan, China), CTSK (1:1,000, Abcam), Cytochrome c (1:1,000, affinity), ERK (1:1,000, Abbkine), IκB-α (1:1,000, Signalwayκ Antibody, Maryland, USA), MMP9 (1:500, ProteinTech, Wuhan, China), NFATc1 (1:1,000, Cell Signaling Technology, Danvers, USA), p-ERK (1:1,000, ProteinTech), and tubulin (1:1,000, affinity) were used to detect the target proteins. After washing for 30 min (5 min * 6 times) with TBST, corresponding secondary antibodies purchased from Abbkine (1:8000) were incubated for 2 h at RT. The images were taken through a Bio-Rad imager after developing the membrane with a HRP substrate. Band intensities were analyzed by the Image J software.
3
Bovine Herpesvirus Type 1 Cell Study
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Bovine herpesvirus type 1 (BHV−1) and MDBK were stored in the cell biology laboratory of Heilongjiang Bayi Agricultural University (Daqing, China). The Ribo FECTTMCP transfection reagent and Ribo lncRNA smart silencer interfering plasmid were designed and synthesized by Guangzhou RiboBio. RNA extraction, cDNA reverse transcription, and fluorescence quantitative PCR kits were all purchased from Nanjing Vazyme Biotechnology. A BCA protein concentration determination kit and RIPA lysate were purchased from Solarbio. Beta actin and sheep anti−mouse IgG−HRP antibodies were purchased from Bioss. NF−κB, JNK and IκB antibodies were purchased from Affinity Biosciences.
4
Western Blot Analysis of Inflammatory Markers
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Retinas and BV2 cells were isolated and lysed with RIPA (Sangon, Shanghai, China) according to the manufacturer’s protocol. Proteins were quantified using the BCA Protein Assay Kit (Beyotime, Shanghai, China). Then the proteins were separated by 10% SDS-page gels and transferred to the PVDF membrane. Primary antibodies used to incubate overnight at 4 °C were as follows: TNFα (1:1000; Abcam, Cambrige, UK), IL-1β (1:1000; Proteintech, Chicago, IL, USA), IL-6 (1:1000; Proteintech, Chicago, IL, USA), iNOS (1:1000; Affinity, USA), NFκB (1:1000; Proteintech, Chicago, IL, USA), p- NFκB (1:1000; Abcam, Cambrige, UK), ERK1/2 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), p-ERK1/2 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), actin (1:1000; Abmart, Shanghai, China) and beta-actin (1:1000; Bioss, Beijing, China). Blots were developed using chemiluminescence (Beyotime, Shanghai, China) and were quantified using Image J software. Band signals was normalized to actin or beta-actin.
5
Western Blot Analysis of Hippocampal Proteins
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On day 27, 8 mice in each group were euthanized by an intraperitoneal injection of pentobarbital sodium (150 mg/kg body weight) to harvest their hippocampi. Right hippocampi were used for WB analysis. After homogenate and protein extraction, SDS-PAGE was performed with a 8% separating gel and a 5% stacking gel and transferred to a 0.45 μm PVDF membrane. Membranes were blocked using 5% non-fat milk in Tris-buffered saline supplemented with 0.1% Tween 20. The primary antibody (PLA2, 1:500, Abcam, United States; COX-1, 1:1000, Abcam, United States; CYP2C23, 1:1000, USBio, United States; beta-actin, 1:500, Bioss, United States) was added, followed by incubation overnight at 4°C. The secondary antibody (goat polyclonal rabbit IgG antibody-HRP, 1:3000, Bioss, United States) was added before shaking and incubation at room temperature for 1 h. HRP-ECL luminous liquid was added, and X-ray film exposure was completed in a dark room following developing and fixing. After calibrating the control markers, the scanning and analysis were performed by Quantity One. All western blot bands were normalized with their corresponding β-actin expression (loading control), for the appropriate evaluation of protein expressions. The relative expression of PLA2, COX-1, and CYP2C23 was compared in each group.
6
Western Blot Analysis of Tight Junction Proteins
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After homogenate and protein extraction, SDS-PAGE was performed with a 8% separating gel and a 5% stacking gel and transferred to a 0.45-μm PVDF membrane. Then, the membranes were incubated with occludin (1:500, Invitrogen, United States), ZO-1 (1:500, Invitrogen, United States), and beta-actin (1:500, Bioss, United States). After washing, the proteins of interest were labeled with the secondary antibody (goat polyclonal rabbit IgG antibody-HRP, 1:3000, Bioss, United States). HRP-ECL luminous liquid was added, and X-ray film exposure was completed in a dark room following development and fixation. All WB bands were normalized with their corresponding β-actin expression for the appropriate evaluation of protein expressions. The relative expression of occludin and ZO-1 was compared in each group.
7
Subcellular Protein Extraction Protocol
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Cytoplasm and nucleus fractions were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Primary antibodies used were as follows: NFκB (1:1000; Proteintech, Chicago, IL, USA), AhR (1:1000; Abclonal, Wuhan, China), beta-actin (1:1000; Bioss, Beijing, China), actin (1:1000; Abmart, Shanghai, China), SP1(1:1000; Abcam, Cambrige, UK) and Histone-H3(1:1000; Proteintech, Chicago, IL, USA). Band signals was normalized to beta-actin /actin (cytoplasm) or SP1/Histone-H3 (nucleus).
8
Quantitative Protein Expression Analysis
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Cell lysates were prepared by using a mammalian cell lysis kit (Sigma, USA). The protein expression by western blotting was repeated thrice for each cell line. The following primary antibodies; polyclonal VEGFA, bFGF, TGFβ1 and E-cadherin (Abcam, UK), beta Actin (Bioss, USA) and EGFR (Santa Cruz, USA) were used. Following the peroxidase-conjugated secondary IgG (H + L) (Jackson Immuno Research, USA), immune complexes were visualized using luminol (Bio-Rad, USA). The chemiluminescence from the specific bands was visualized by Molecular Imager GelDoc XR+ ChemiDoc XRS+ Imaging System (BioRad, USA). The expression ratios of VEGFA, E-cadherin, TGFβ1, EGFR, bFGF, and β-actin from the blots were analyzed using Molecular Imager GelDoc XR+ ChemiDoc XRS+ Imaging System and Image Lab Software 6.0.1 (Bio-Rad, USA). After determining the intensities of the obtained bands, normalized protein fold ratios were calculated by proportioning the intensity value of each band to the corresponding β-actin band for each dose level.
9
Protein Expression Analysis in Frozen Kidney
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Frozen kidney tissues were lysed and centrifuged to obtain the supernatant, followed by proteins separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We transferred proteins to nitrocellulose (NC) membranes and probed the membranes with primary antibodies against the following proteins overnight at 4 °C, including beta-actin (Beijing Biosynthesis Biotechnology Co. 0061R; 1:2000), Pax2 (Proteintech, 21385; 1:800), vimentin (Abcam ab8069; 1:1,000), Kim1(R&D Systems, AF1817; 1:500), ERK1/2 (CST, #9102; 1:800), p-ERK1/2 (CST, #9101; 1:800), cyclin D1 (Proteintech 60186; 1:1,000), and cyclin E1 (Proteintech 11554; 1:1,000). Finally, blots were probed with horseradish peroxidase-conjugated IgG (Santa Cruz Biotechnology, sc-2096; 1:1000), with bands visualized by enhanced chemiluminescence, and quantified densitometrically using the Quantity One software (Bio-Rad Laboratories).
10
Kidney Tissue Protein Analysis Protocol
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The frozen kidney tissues were lysed with radioimmunoprecipitation assay lysis buffer and centrifuged at 12,000g at 4 C for 30 minutes to obtain the supernatant. Equal amounts of proteins were obtained from each sample and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membranes and probed with the primary antibodies against the following proteins at 4 C overnight: beta-actin (0061R, 1:2000; Beijing Biosynthesis Biotechnology Co., Beijing, China), cleaved caspase-3 (9664; 1:500; Cell Signaling Technology, Boston, MA), Bax (2772, 1:1000; Cell Signaling Technology), Bcl2 (2876, 1:1000; Cell Signaling Technology), cytochrome C (4280, 1:1000; Cell Signaling Technology), LC3 (L7543, 1:2000; Sigma, Los Angeles, CA), Atg5 (108327, 1:1000; Abcam), Beclin1 (B6186, 1:1000; Sigma), P62 (96416; 1:1000; Abcam), IL1b (E1713, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), IL6 (12912, 1:500; Cell Signaling Technology), p-nuclear factor kB (3033, 1:1000; Cell Signaling Technology), or nuclear factor kB (8242, 1:1000; Cell Signaling Technology). The blots were probed with horseradish-peroxidase-conjugated IgG (sc-2096, sc-2963, 1:1000; Santa Cruz Biotechnology). Immunoreactive bands were visualized by enhanced chemiluminescence, and densitometry was performed in Quantity One software (Bio-Rad Laboratories, Hercules, CA).
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