For western blotting (WB) analysis, resuspended mPFC homogenates (alcohol group n = 4, air group n = 4) were loaded at 20 μg measured with Bradford protein assay (Bio-Rad, Hercules, CA, United States). Proteins were separated on a 4–12% Nu-Page Bis-Tris gel in MOPS buffer (Invitrogen, Carlsbad, CA, United States) at 140 V for 2 h followed by transfer to a PVDF membrane (Invitrogen) at 30 V for 1 h. The membrane was blocked in 5% non-fat milk for 1.5 h. Samples were then immunoblotted overnight at 4°C (5% BSA in 1 × TBST) with antibodies specific to Fis1 (1:500; Abcam, #ab71498), Mfn2 (1:500; Cell Signaling Technology, #9482), Drp1 (1:200; Santa Cruz Biotechnology, #sc-271583, Dallas, TX, United States), OPA1(1:200; Santa Cruz Biotechnology, #sc-393296, Dallas, TX, United States), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000; #MAB374, Millipore). Following three 10-min washes (1 × TBST), immunoblots were incubated (5% BSA) for 1 h at room temperature with respective anti-rabbit and anti-mouse secondary antibodies (1:1000, Millipore). Blots were visualized with the Radiance Q Chemiluminescent Substrate (Azure Biosystems, Dublin, CA, United States), developed on an Azure 300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, Dublin, CA, United States), and band optical density quantification was performed using NIH ImageJ software.
Anti rabbit and anti mouse secondary antibodies
Manufactured by Merck Group
Sourced in United States
Anti-rabbit and anti-mouse secondary antibodies are laboratory reagents used in various immunoassays and immunochemical techniques. These antibodies are designed to bind to primary antibodies that have been raised in rabbit or mouse hosts, respectively, allowing for the detection and visualization of target proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti inos, by Santa Cruz Biotechnology (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Anti cox 2, by Santa Cruz Biotechnology (2 mentions)
Anti ho 1, by Santa Cruz Biotechnology (2 mentions)
Anti p65, by Santa Cruz Biotechnology (2 mentions)
Mops buffer, by Thermo Fisher Scientific (1 mentions)
Radiance q chemiluminescent substrate, by Azure Biosystems (1 mentions)
Azure 300 chemiluminescent western blot imaging system, by Azure Biosystems (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Anti proliferating cell nuclear antigen, by Santa Cruz Biotechnology (1 mentions)
Anti nrf2, by Santa Cruz Biotechnology (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Anti p p38, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti p jnk, by Cell Signaling Technology (1 mentions)
10 protocols using anti rabbit and anti mouse secondary antibodies
1
Mitochondrial Dynamics Alterations in Alcohol Exposure
2
Phytochemical Analysis of C. tricuspidata
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Tissue culture reagents, such as Roswell Park Memorial Institute 1640 (RPMI 1640), Dulbecco’s Modified Eagle’s Medium (DMEM), and fetal bovine serum (FBS), were purchased from Gibco BRL Co. (Grand Island, NY, USA). All chemicals were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Primary antibodies, including anti-HO-1, anti-Nrf2, anti-p65, anti-COX-2, anti-iNOS, anti-β-actin, and anti-proliferating cell nuclear antigen (PCNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-rabbit and anti-mouse secondary antibodies were purchased from Millipore (Billerica, MA, USA). ELISA kits for PGE2, IL-6, and TNF-α were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). The structure of each of the sixteen C. tricuspidata compounds was determined by analyzing various spectroscopic data, including mass spectroscopy and nuclear magnetic resonance (NMR) data, which were consistent with what had been previously reported [10 (link)].
3
Detailed Anti-inflammatory Compound Isolation
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Roswell Park Memorial Institute 1640 (RPMI 1640) and fetal bovine serum were purchased from Gibco BRL Co. (Grand Island, NY, USA). All chemicals were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The primary antibodies anti-iNOS, anti-β-actin, anti-p65 and anti-HO-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and anti-rabbit and anti-mouse secondary antibodies from Millipore (Billerica, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for PGE2, IL-6, and TNF-α were purchased from R&D Systems Inc. (Minneapolis, MN, USA). The isolation and structural determination of the 11 compounds from Morus alba have been described elsewhere [19 (link),20 (link),21 (link)].
4
Isolation and Structural Elucidation of Bioactive Constituents
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The materials and equipment we used for the isolation and structure determination of constituents are described in a previous study [16 (link)]. Tissue culture reagents, such as Roswell Park Memorial Institute 1640 (RPMI1640) and fetal bovine serum, were purchased from Gibco BRL Co. (Grand Island, NY, USA). All chemicals were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Primary antibodies, including anti-iNOS, anti-COX-2, and anti-β-actin, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-p-JNK, anti-JNK, anti-p-ERK, anti-ERK, anti-p-p38, and anti-p38 were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-rabbit and anti-mouse secondary antibodies were purchased from Millipore (Billerica, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for PGE2, IL-6, and TNF-α assessments were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). The compound structure was determined by analyzing various spectroscopic data, including mass spectroscopy and nuclear magnetic resonance, which were consistent with those previously reported [17 (link)].
5
Quantitative Protein Analysis of HLSC
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Total protein was extracted from HLSC differentiated in RCCS, from human hepatocytes (Lonza) or mouse primary hepatocytes using lysis buffer containing 1% Triton X-100 supplemented with protease inhibitors (Complete Mini, Roche) and 50 microgram samples were separated by 4–15% SDS-PAGE (Biorad). After protein transfer, nitrocellulose membrane was saturated with 5%BSA and incubated overnight at 4 °C with rabbit anti-Ugt1A antibody (Ugt1A, sc-25847, Santa Cruz Biotechnology) and mouse anti-vimentin (in-house). After washing, the membrane was incubated for one hour at room temperature with anti-rabbit and anti-mouse secondary antibodies from Sigma and developed using ECL (Biorad) on Chemidoc imaging system (Biorad). Densitometric analysis was performed using the volume analysis tool of ImageLab software (Biorad Laboratories Inc).
6
Characterization of MDA-MB-231 Breast Cancer Cells
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The MDA-MB-231 (MDA231) breast cancer cell line (cat#HTB-26) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Human mammary epithelial cells (HMECs) (cat#A10565) were procured from Thermo Fisher Scientific (Waltham, MA, USA). Fluorescent-labeled antibodies for flow cytometry experiments, including GAPDH (cat#AF5718); ICAM (cat#BBA20); PECAM (cat#FAB3567P); integrin α5 (cat#FAB1864P); and EpCAM (cat#FAB9601P) were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies for immunoblotting—P53 (cat#2527), pRb (S807) (cat#8516), Chk1 (cat#2348), cleaved caspase-3 (cat#9664), and β-actin (cat#3700 and cat#8457)—were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-rabbit and anti-mouse secondary antibodies (cat#21537 and 21538M) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protease inhibitor cocktail (cat#P8340) was purchased from Millipore Sigma (Burlington, MA, USA).
7
Protein Expression Analysis by Western Blot
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The cells were seeded on Petri dishes and induced as described previously. After 72 h, the cells were detached and the protein was isolated with RIPA buffer (Sigma-Aldrich, Saint Louis, MO, USA) as described previously [12 (link)]. Membranes were blocked in 5% fat-free milk in TBST buffer prior to overnight incubation in 4 °C in the primary antibodies anti- MMP-2 (1:200 in 1% fat-free milk, SantaCruz Biotechnology, Dallas, TX, USA) or anti-GAPDH (1:1000, SantaCruz Biotechnology, Dallas, TX, USA) as a reference. After incubation, the membranes were washed three times with TBST buffer and incubated with anti-rabbit and anti-mouse secondary antibodies (1:15,000, Sigma-Aldrich, Saint Louis, MO, USA) for MMP-2 and GAPDH, respectively, for four hours at 4 °C. The membranes were washed once again and the bands were visualized with using Novex® AP Chromogenic Substrate (BCIP/NBT) (Thermo Fisher Scientific, Inc, Waltham, MA, USA). Densitometric analysis was conducted with ImageJ [36 ] (National Institutes of Health, Bethesda, MD, USA). The experiment was conducted in triplicate.
8
Western Blot Analysis of Signaling Proteins
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30 μg of protein were separated on polyacrylamide gels (Lonza), transferred to nitrocellulose membranes (Invitrogen) and blocked for 1 hour in blocking solution (2.5% milk powder (Biorad) in PBS containing 0.1% Tween-20 (PBS-T)). Membranes were incubated at 4°C overnight with primary antibody at a 1:1000 dilution in blocking solution (unless otherwise stated) against p-HER2tyr1221/1222, p-EGFRtyr1173, AKT, p-AKTser473, ERK, pERKthr202/tyr204, eEF2, p-eEF2thr56, mTOR, p-mTORser2448, eEF2k, p-eEF2kser366 (Cell Signaling Technology), p-eEF2kser359 (1:200) (SantaCruzBiotechnology), HER2 (Calbiochem), EGFR (1:250) (Neomarkers), and α-tubulin, anti-mouse and anti-rabbit secondary antibodies (Sigma-Aldrich). Detection was performed using Luminol (SantaCruzBiotechnology) or ECL Advance (GE Healthcare).
9
DMBA-Induced Cancer Cell Signaling
10
Noradrenaline Binding and Receptor Assay
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Notably, 1-[7,8 3H]-noradrenaline (specific activity 39 Ci mmol-1), was from Amersham Radiochemical Centre (Buckinghamshire, UK). The ellagic acid was from Sigma Aldrich (Mi., Italy). 6-Nitroquipazine maleate was donated from Duphar, Amsterdam, the Netherlands. 1-(2-(Bis-(4-fluorophenyl) methoxy) ethyl)—4-(3-phenylpropyl) piperazine dihydrochloride (GBR12909) was purchased from Tocris Bioscience (Bristol, UK). Anti-mouse and anti-rabbit secondary antibodies were from Sigma-Aldrich (USA). The SuperScript® VILO™ cDNA Synthesis Kit was from Life Technologies (USA).
This could account for the effects here described, allowing the conclusion that either EA or UROs could have a role in determining their onset.
This could account for the effects here described, allowing the conclusion that either EA or UROs could have a role in determining their onset.
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