Bamboo-leaf flavonoids (the mass fraction of four carbon glycoside flavonoids was 80.66%, Supplementary Figure S2 ) were prepared in laboratory. L-α-Phosphatidylcholine, cholesterin, CH, AL, 2–2′azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azobis (2-methylpropionamidine) dihydrochloride (AAPH), 3-(4,5-dimthyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma Aldrich (St. Louis, MO, USA). Reagents required for cell culture were purchased from HyClone (Logan, UT, USA) and Gibco (Grand Island, NY, USA), respectively. K9M-H3 and Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (H + L) antibodies were purchased from ABclonal Technology (Wuhan, Hubei, China); p21, p16 and Goat Anti-Rabbit secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). HPLC-grade methanol was obtained from TEDIA (Cincinnati, OH, USA). The 10 kDa dialysis bag was provided by Spectrum (San Jose, CA, USA). Other chemicals and reagents were purchased from Sinopharm (Shanghai, China).
Goat anti rabbit secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States, China
Goat anti-rabbit secondary antibody is a laboratory reagent that is used to detect the presence of rabbit primary antibodies in various immunoassays, such as Western blotting, ELISA, and immunohistochemistry. It is a polyclonal antibody produced in goats and specifically binds to the Fc region of rabbit antibodies, allowing for the amplification and visualization of the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Anti gapdh, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Goat anti mouse secondary antibody, by Cell Signaling Technology (4 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
β actin, by Proteintech (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Horse anti mouse secondary antibody, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Nupage mini gel, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Anti sirt1, by Cell Signaling Technology (2 mentions)
Hplc grade methanol, by Tedia (1 mentions)
65 protocols using goat anti rabbit secondary antibody
1
Bamboo-Leaf Flavonoid Extraction and Antioxidant Evaluation
2
Quantitative Analysis of α-Synuclein and TH Phosphorylation
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Western blot analysis was performed to examine the phosphorylation of α-synuclein (Ser129), that of TH (Ser40), and β-actin levels. Proteins in samples (5 μg) were electrophoresed on sodium dodecyl sulfate-poly acrylamide gels (SDS-PAG, 10–15%) and they were transferred to a membrane of polyvinylidene difluoride. The blots were incubated overnight at 4°C in primary antibodies (rabbit source) against α-synuclein, phosphorylated α-synuclein (at Ser129) (p-α-synuclein), TH, phosphorylated TH (at Ser40) (p-TH), and β-actin were purchased from the Cell Signaling Technology Inc. (Beverly, MA, USA) (dilutions, 1:1000) in Tris-buffered saline containing Tween-20 (TBS-T) and bovine serum albumin (BSA, 5%). Then the blots were kept in goat anti-rabbit secondary antibodies (1:5000 in TBS-T containing 5% BSA) (Cell Signaling Technology Inc.) for 1 hour at room temperature in accordance with the standard procedure.
After washes, the transferred proteins were incubated with the substrate solution of electrochemiluminescence (ECL) (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in accordance with the instructions of the manufacturer. Reactive bands were visualized on radiographic film and digitalized using MP Navigator EX 2.0 (Canon, Tokyo, Japan).
After washes, the transferred proteins were incubated with the substrate solution of electrochemiluminescence (ECL) (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in accordance with the instructions of the manufacturer. Reactive bands were visualized on radiographic film and digitalized using MP Navigator EX 2.0 (Canon, Tokyo, Japan).
3
Molecular Mechanisms of TGF-β1 and TNFα
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Recombinant human TGF-β1 and TNFα were purchased from R&D Systems (Minneapolis, MN). Lithium chloride (LiCl) was purchased from Sigma-Aldrich (St. Louis, MO) and SB216763 from Selleckchem.com (Houston, TX). Antibodies to E-Cadherin (E-cad), β-actin, GAPDH, Smad3, phosphor-Smad3 (Ser423/425), Akt, phospho-Akt (Ser473), GSK3β, phospho-GSK3β (Ser9), peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Cell Signaling (Danvers, MA). Antibody to alpha-smooth muscle actin (α-SMA) was purchased from Sigma Aldrich (St. Louis, MO). Antibodies to fibronectin extra domain A (FN-EDA) and type-1 collagen (col-1) were purchased from Abcam (Cambridge, MA). Antibody to BMAL1 was purchased from Novus (Littleton CO). Acetyl-BMAL1 (K538) antibodies were from EMD Millipore (Billerica, MA) and Ameritech Biomedicines (Houston, TX). Plasminogen activator inhibitor type 1 (PAI1) antibody was obtained from Peprotech (Rocky Hill, NJ).
4
Antibody Sources for Histone and Protein Analysis
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P21(AB109199) was purchased from Abcam; H3K4me3 (AF5704), p21(AF5252), p53 (AF0255), UB (AF0306), and phospho‐histone H2AX (Ser139) (AF5836) antibodies were purchased from Shanghai Beyotime Biotechnology; HDAC1 (D5C6U), goat anti‐mouse secondary antibodies, goat anti‐rabbit secondary antibodies, and immunofluorescence fluorescent secondary antibodies were purchased from Cell Signaling Technology. DA was purchased from MedChemExpress (https://www.medchemexpress.cn/ ).
5
Antioxidant and Neuroprotective Assays
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Chemicals and reagents were purchased as follows: L-glutamic acid, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, curcumin, dimethyl sulfoxide (DMSO), Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), and N-acetyl cysteine (NAC) from Sigma–Aldrich (St. Louis, MO, USA); 10× penicillin-streptomycin from Hyclone (UT, USA); Bradford reagent from Bio-Rad (Hercules, CA, USA); 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) from Bio Basic (Markham, ON, Canada); gallic acid from TCI America (Portland, OR, USA); L-ascorbic acid from Calbiochem (San Diego, CA, USA); 2′, 7′-dichlorodihydro fluorescein diacetate (H2DCFDA) from Molecular Probes (Eugene, OR, USA); trypsin-EDTA (0.25%) and penicillin/streptomycin solution from Gibco (Waltham, MA, USA); primary antibodies against p44/42 kinase (ERK), phospho-p44/42 (p-ERK), apoptotic-inducing factor (AIF), β-actin, Lamin B1, and goat anti-rabbit secondary antibodies from Cell Signaling Technology (Danvers, MA, USA); primary antibodies against brain-derived neurotrophic factor (BDNF) from Abcam (Cambridge, UK); the annexin V- Fluorescein isothiocyanate (FITC) apoptosis detection kit from BioLegend (San Diego, CA, USA); and the NE-PER nuclear and cytoplasmic extraction reagents from Thermo Scientific (Rockford, IL, USA).
6
Investigating Inflammatory Pathways
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CGA (#C3878) was purchased from Sigma-Aldrich (St. Louis, United States ). CGA was resolved in dimethyl sulfoxide and then sterile filtered before used and dissolved in distilled water. Rabbit-anti-mouse polyclonal primary antibodies against Toll like receptor 4 (TLR4), nuclear transcription factor-κB (NF-κB), p-NF-κB, inhibitor of NF-κB-α (IκB-α), p-IκB-α, β-actin, zonula occludens-1 (ZO-1), and Occludinwere purchased from Proteintech group. Goat-anti-rabbit secondary antibodies were purchased from Cell Signaling Technology.
7
Protein Extraction and Western Blot Analysis
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Cells grown on 6-well plates were washed with PBS and lysed with RIPA lysis buffer (Boston Bioproduct; Cat# BP-115) containing a cocktail of protease and phosphatase inhibitor (Thermo Scientific; Cat# 1861280) at 4 °C for 25 min to extract protein. Cell extracts were resolved by SDS/PAGE (Bio-Rad, Hercules, CA, USA; Cat# 5671094) and transferred to PVDF membranes (Cytiva Amersham, Fisher Scientific; Cat# 10600023). The resulting membranes were incubated with primary antibodies for 16 to 18 h at 4 °C, washed with 1× TBST (Boston Bioproducts, Inc., Ashland, MA, USA; Cat# IBB-580X), and incubated for 1 h with goat anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA; Cat# 7074) at room temperature prior to revelation using chemiluminescence (Thermo Scientific; Cat# 34580).
8
Western Blot Analysis of Liver Proteins
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Liver tissue was homogenized in RIPA lysis buffer and used for Western blot analysis. Briefly, 40 μg of protein extracts were boiled with sodium dodecyl sulfate sample buffer for 5 minutes before being electrophoretically resolved on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Pierce Chemical Company, Rockford, Illinois, USA). The membranes were then blocked for 1 hour at room temperature in 5% skimmed milk containing 1× Tris-buffered saline and 0.1% Tween 20. After blocking, the membranes were incubated overnight at 4°C with rabbit-anti-rat monoclonal antibodies (1:500 dilution; Cell Signaling, Boston, MA, USA). The membranes were incubated for 2 hours with goat-anti-rabbit secondary antibodies (1:2,000 dilution; Cell Signaling). Finally, the membranes were washed and an electrochemiluminescence (ECL) signal detection kit (Amersham/GE Healthcare, Piscataway, NJ, USA) was used for visualization of the protein bands.
9
Western Blot Analysis of Cellular Proteins
10
Chrysanthemum coronarium Leaf Assay
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Chrysanthemum. coronarium L. leaves were purchased from a local market (Jeonju, Republic of Korea). AD, methanol (MeOH), and ethanol were obtained from Sigma-Aldrich (St. Louis, MO, USA). Radioimmunoprecipitation assay buffer was purchased from Beyotime (Los Altos, CA, USA). BCA Protein Assay Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). SDS-PAGE gels and polyvinylidene fluoride membranes (PVDF) were obtained from Millipore (Burlington, MA, USA). TRIzol reagent and SYBR Green PCR Master Mix Kit were obtained from Invitrogen (Waltham, CA, USA). ICAM-1, COX-2, α-SMA, and β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Goat anti-rabbit secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). iScript cDNA synthesis kit was purchased from Bio-Rad (Hercules, CA, USA). BUN, SCr, calcium, and phosphorous kits were purchased from Chema Diagnostica Di Fiore Marco (Monsano, Italy).
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