Cell lysates were prepared in buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 mM sodium pyrophosphate, 1 mM NaF, 1 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM PMSF, 2 μg/ml leupeptin and 10 μg/ml aprotinin (Sigma Aldrich). Western blot was carried out as we have previously published [49 (link)]. The primary and secondary antibodies used were: anti-PCSK9 (1:1000; Abcam, CA, USA); anti-actin (1:1,000; Santa Cruz Biotechnology), anti-mouse (1:3,000; GE Healthcare) and anti-rabbit (1:3,000).
Anti rabbit
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Denmark
Anti-rabbit is a laboratory reagent used to detect and quantify the presence of rabbit-derived proteins or antibodies in various samples. It serves as a tool for immunoassays, Western blotting, and other analytical techniques requiring the specific recognition of rabbit-derived biological molecules.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse, by GE Healthcare (17 mentions)
Anti goat, by Santa Cruz Biotechnology (8 mentions)
Imagequant tl, by GE Healthcare (6 mentions)
Anti mouse, by Agilent Technologies (5 mentions)
Ecl prime, by GE Healthcare (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Anti actin, by Santa Cruz Biotechnology (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Hrp conjugated anti mouse, by GE Healthcare (3 mentions)
Anti mouse, by Bio-Rad (3 mentions)
Ecl prime detection kit, by GE Healthcare (3 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Ec3 imaging system, by Analytik Jena (2 mentions)
Anti mmp 13, by Santa Cruz Biotechnology (2 mentions)
Anti nos2, by Cell Signaling Technology (2 mentions)
Anti iκbα, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated anti mouse, by GE Healthcare (2 mentions)
Novex tris glycine sds page, by Thermo Fisher Scientific (2 mentions)
61 protocols using anti rabbit
1
Quantitative Western Blot Analysis of PCSK9
2
Whole Cell Protein Extraction and Immunoblotting
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Whole cell protein extraction was developed using a lysis buffer. Electrophoresis and blotting procedures have been described previously13 (link). Immunoblots were incubated with the appropriate antibody (anti-phospho p38 diluted 1:1000, Millipore, MA, USA; anti-p38 diluted 1:1000, Millipore, MA, USA; anti IκBα diluted 1:1000, Cell Signaling Technology, MA, USA; anti-MMP-13 diluted 1:500, Santa Cruz Biotechnology, CA, USA; anti-NOS2 diluted 1:1000, Cell Signaling Technology, MA, USA; anti-COX-2 diluted 1:1000, Dako, Denmark) and visualized using an Immobilon Western kit (Millipore, MA, USA) and anti-rabbit (GE Healthcare, UK) horseradish-peroxidise-labelled secondary antibody diluted 1:2000. To confirm equal loading for each sample, after stripping in glycine buffer at pH3, membranes were reblotted with anti-β-actin antibody diluted 1:5000 (Sigma, MO, USA). Autoradiographs were analyzed with an EC3 imaging system (UVP, CA, USA).
3
Western Blot Analysis of Amyloid Proteins
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Proteins (10 μg) from cell lysates or culture media (20 μl) were heated for 10 min at 70°C in loading buffer (lysis buffer supplemented with 0.5 M DTT and staining Nupage blueTM), separated in 4–12% NupageTM bis-Tris gel and transferred for 2 h at 30 V onto nitrocellulose membranes. Ponceau Red staining was used to check gel loading and transfer accuracy. After blocking (5% non-fat milk in PBS), membranes were incubated overnight at 4°C with the primary antibodies: anti-Amyloid β Antibody, clone W0-2 (1/2,500), Anti-Amyloid Precursor Protein, C-terminal antibody (1/2,000), anti-GLuc antibody (1/2,000). Membranes were washed with PBS-Tween (0.005%) and incubated with the secondary antibodies anti-mouse (1:10,000) or anti-rabbit (1:10,000) coupled to peroxidase prior to ECL detection from GE Healthcare (Little Chalfont, UK). Signals were quantified with a Gel Doc 2000 imaging system coupled to Quantity oneTM software from Bio-Rad (Hercules, CA, USA).
4
Western Blot Analysis of Cellular Signaling
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Aliquots of treated cells as indicated were treated with whole-cell lysis buffer (10 mM Tris-HCl, 250 mM sodium chloride, 50 mM sodium fluoride, 0.5% Triton X-100, 0.1% sodium dodecyl sulfate, 10% glycerol, 1 complete pill of protease inhibitor mixture (Roche), 1 mM phenylmethysulfonyl fluoride, 100μM sodium orthovandate, 2mM iodoacetic acid, and 5 mM ZnCl2). Protein concentration was determined by Thermo Scientific Pierce BCA Protein Assay Kit following supplier stipulations. Equal amounts of protein for each sample were separated on a 4 to 12% Bis-Tris precast acrylamide gel (BioRad) and transferred to a nitrocellulose membrane. Western blots were probed with antibodies against the following antigens: Actin, Bip/GRP78, SMAD2/3, SMAD2/3 S423 and p27KIP (Santa Cruz Biotechnology); 4E-BP1, 4E-BP1 Thr37/46, p70S6K Thr 389, ERK Thr202/204, FAKTyr861 and Y397, Src Tyr416 AMPK Thr172 (Cell Signaling); p21 (CIP1/WAF) (Neomarkers); LC3 I-II antibody (Novus Biologicals). The secondary antibodies were horseradish-peroxidase–conjugated anti-mouse (GE Healthcare), anti-rabbit (GE Healthcare) or anti-goat (Santa Cruz Biotechnology). Detection was performed by an enhanced chemiluminescent method (Immobilon Western, Millipore). Protein bands were quantified using ImagesJ 1.44p (Wayne Rasband, NIH) software and normalized to Actin.
5
Melanoma Marker Protein Expression
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Changes in protein expression of the melanoma cell differentiation markers tyrosinase and gp100 were assessed with western blotting using standard conditions. The primary antibodies used were rabbit anti-tyrosinase (Abcam; Cambridge, UK), anti-gp100 (Abcam; Cambridge, UK), anti-P-ERK1/2, anti-ERK1/2 (Cell signaling Technology; Danvers, MA, USA), and mouse anti-Hsp70 (Stressgen Bioreargents; Michigan, USA) and anti-β actin (Chemicon; Hampshire, UK). The secondary antibodies used were anti-mouse (Dako A/S, Glostrup, Denmark) and anti-rabbit (GE Healthcare Life Sciences; Buckinghamshire, UK). Antibody-target protein interactions were visualised by enhanced chemiluminescence (GE Healthcare Life Sciences).
6
Protein Expression Analysis in Muscle Tissue
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Muscle homogenates were prepared by grinding tissue with ice-cold lysis buffer cell extraction buffer (Invitrogen) as previously described (13 (link)). Protein concentrations were measured using a BCA protein assay (Thermo Scientific). Proteins were separated by 4–20% Novex Tris-Glycine SDS-PAGE (Invitrogen) and transferred to a PVDF membrane (Biorad). Membranes were incubated overnight with anti-FABP3 (Lifespan Biosciences, Seattle, WA; LS-C138955), anti-CPT1B (Abcam, Cambridge, MA; ab15703) or anti-actin (Santa Cruz, Dallas, TX; sc-1616), followed by incubation with appropriate secondary horseradish peroxidase-conjugated antibodies, anti-rabbit (GE Healthcare, Piscataway, NJ; NA931) and anti-goat (Santa Cruz, sc-2020). Immunoreactive proteins were visualized by chemiluminescence reagent (ECL Prime; GE Healthcare) and quantified by densitometric analysis using ImageQuantTL (GE Healthcare).
7
STING Signaling Pathway Analysis
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Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor (Cat. 25765800, Sigma Aldrich) and a phosphatase inhibitor (Cat. P5726, Sigma Aldrich). After denaturing the samples at 95°C for 5 minutes, 30μg of each protein sample was separated using SDS-PAGE and transferred onto nitrocellulose membranes (Cat. 1620112, Bio-Rad, Hercules, CA). Next, the membranes were blocked with 5% BSA for 1 hour, and then incubated with primary antibodies against STING (Cat. 13647S, Cell Signaling), IRF3 (Cat. 4302S, Cell Signaling), p-IRF3 (Cat. 4947S, Cell Signaling), TBK1 (Cat. 3013S, Cell Signaling), p-TBK1 (Cat. 5483S, Cell Signaling), β-actin (Cat. 3700S, Cell Signaling), and Vinculin (Cat. 13901S, Cell Signaling) overnight at 4°C. The following day, the membranes were washed with PBST and incubated with anti-mouse (Cat. NXA931, GE Healthcare, Chicago, IL) or anti-rabbit (Cat. NA934V, GE Healthcare) secondary antibody for 2 hours before developing with ECL substrates (Cat. 170506, BioRad). The gel images were captured using Chem-DocXRS image acquisition machine (Bio-Rad).
8
Western Blot Analysis of Adipose Tissue
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BAT, iWAT, and gWAT depots were homogenized with 300–500 ul of RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.25% deoxycholate) containing protease inhibitor cocktail (Roche) and phospho-STOP cocktail (Roche), followed by pelleting of the insoluble debris at 13,000×g for 15 min at 4 °C. The protein concentrations of lysates were determined by BCA assay (Thermo Scientific), and 30 μg of lysate was separated by Tris-glycine SDS-PAGE (10% polyacrylamide). Proteins were transferred to nitrocellulose membranes (Protran BA 83, Whatman), blocked in 3% BSA in 1X TBST (Tris-buffered saline with Tween 20), and incubated with primary antibodies overnight. The blots were probed with the following antibodies: Ucp1 (Sigma; U6382), Ndufb8, Sdhb, Uqcrc2, Atp5a (MitoProfile total OXPHOS, Abcam; ab110413), Aco2 (Cell Signaling; 6922), Mcad (GeneTex; GTX32421), Pcx (Abcam; ab128952), Vdac (Calbiochem; PC548), Pdh E2/E3bp (Abcam; ab110333), Acot2 (Sigma; SAB2100030), beta-Actin (Sigma; A2228), and Hsc-70 (Santa Cruz; sc-7298). Cy3-conjugated anti-mouse (Invitrogen), or HRP-conjugated anti-mouse (GE Healthcare) or anti-rabbit (GE Healthcare) secondary antibodies were used appropriately. Images were collected and analyzed using an Alpha Innotech FluorChemQ.
9
Immunoprecipitation and Western Blotting Analysis of Transfected Proteins
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Transfected HEK293T or HeLa cells were harvested from 100 mm dishes 48 h post-transfection and lysed in IP buffer (50 mM Tris-HCl pH 7.5, 150–300 mM NaCl, 1mM Dithiotreitol (DTT) and 0.5% Nonidet P-40) plus protease inhibitors (Roche Molecular Biochemicals). For immunoprecipitation of the transiently expressed Flag-tagged proteins, cell extracts were incubated with 30 μl anti-Flag M2 affinity gel (Sigma) for 4 h at 4°C and the immunopurified proteins were analyzed by western blotting. Antibodies used for other immunoprecipitations or western blotting analysis were anti-Flag rabbit polyclonal antibody (Sigma), anti-Myc (9E10) mAb, anti-human MIZ-1 goat polyclonal antibody (R & D Systems), anti-EBNA3A sheep polyclonal antibody (Exalpha biologicals Inc), anti-Nucleophosmin mAb (Invitrogen) and anti-α-tubulin mAb (B-5–1–2, Sigma). The appropriate anti-mouse (GE Healthcare), anti-rabbit (GE Healthcare), anti-goat (Santa Cruz Biotechnology, Inc) or anti-sheep (Abcam) horseradish peroxidase (HRP)-conjugated antibodies were used as secondary antibodies. Myc-tagged proteins were also revealed using Myc (9E10) HRP-conjugated antibody (Santa Cruz Biotechnology, Inc). Western blots were revealed using Enhanced Chemiluminescence (ECL) (Pierce).
10
Quantification of Extracellular Matrix Proteins
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Aliquots of frozen eWAT (∼150 mg) were homogenized in 200 μl of a buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 2 mM sodium EDTA, 1% NP-40) supplemented with a protease inhibitor cocktail (Sigma). Protein, 30 μg. was subjected to 3 to 8% SDS-PAGE for immunoblot analyses of collagen Ⅰ(Col1), Col3, and Col6 or 10% SDS-PAGE for immunoblot analyses of Acta2, Timp1, and HMGB1. After the membranes were blocked for 30 min in a blocking one solution (Nacalai Tesque), they were incubated with primary antibodies for Timp1 (R&D systems), Col1 (Bioss), Col3 (Bioss), Col6 (Novus Biologicals), Acta2 (abcam), or HMGB1 (abcam) overnight at 4°C. The membranes were incubated with anti-rabbit (GE healthcare) or anti-goat (Proteintech) IgG horseradish peroxidase–conjugated secondary antibody for 1 h in TBS-T solution at room temperature. An enhanced chemiluminescent substrate ECL prime (GE Healthcare) was used to visualize the horseradish peroxidase. Image Quant Las 4000mini (Cytiva) was used to detect the enhanced chemiluminescence. All membranes were stained with Ponceau S after protein transfer for protein normalization.
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