DC or HepG2 cells were pretreated with inhibitors in serum-free media for 30 min at 37 °C. Without washing, fluorescently-labeled protein was added at 10 μg/ml for 1 hr at 37 °C. After washing, MFI of internalized protein was quantitated by flow cytometry. Percent inhibition was calculated as: [(MFI of untreated cells) – (MFI of treated cells)]/(MFI of untreated cells) × 100 %. The following blocking antibodies were used: CD206 (clone 15–2; BioLegend), DC-SIGN (clone 120507), SR-A1 (clone 351620), LOX-1 (clone 331212; all from R&D Systems), CD36 (clone 185-1G2; NeoMarkers), and SR-B1 (rabbit polyclonal; Novus Biologicals). Dimethylamiloride (DMA; 100 μM), mannan (300 μg/ml), and polyinosinic acid (Poly I; 50 μg/ml) were purchased from Sigma.
Sr b1
Manufactured by Novus Biologicals
Sourced in United States
The SR-B1 is a laboratory equipment product. It serves as a core function for specific applications, but a detailed unbiased description cannot be provided while maintaining conciseness and avoiding interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Cd206 clone 15 2, by BioLegend (1 mentions)
Dimethylamiloride dma, by Merck Group (1 mentions)
Polyinosinic acid poly 1, by Merck Group (1 mentions)
Sr a1, by R&D Systems (1 mentions)
Tnf α, by Beckman Coulter (1 mentions)
Dc sign, by BD (1 mentions)
Lox 1, by BioLegend (1 mentions)
Hla abc, by BioLegend (1 mentions)
Cd206, by BD (1 mentions)
Hla dr, by Beckman Coulter (1 mentions)
Accuri c6 cytometer, by BD (1 mentions)
Interleukin 2 (il 2), by BD (1 mentions)
Abcg1, by Novus Biologicals (1 mentions)
Gapdh, by Novus Biologicals (1 mentions)
Wortmannin, by Cell Signaling Technology (1 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 633 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Human apoai, by Merck Group (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Mcp 1, by R&D Systems (1 mentions)
7 protocols using sr b1
1
Receptor-mediated Protein Internalization Assay
2
Endocytic receptor and immune cell analysis
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Endocytic receptors were stained using the following antibodies: CD206/MR (eBioscience), DC-SIGN, CD36 (both from BD Biosciences), SR-A1 (R&D Systems), LOX-1 (BioLegend), and SR-B1 (Novus Biologicals). DC and T cell phenotypes were examined using antibodies against the following markers: HLA-ABC (BioLegend), CD206, CD40, CD80, CD83, IL-2 (BD Biosciences), CD4, CD8, TNF-α, IL-2, and HLA-DR (Beckman Coulter). Data were acquired with an Accuri C6 cytometer (BD Biosciences) and analyzed using CFlow Plus software.
3
Antibody-Based Analysis of Cholesterol Transporters
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Antibodies against ABCA1(Cat#NB100–2068, RRID: AB_535487), ABCG1(Cat# NB400-132SS, RRID: AB_5920491), SR-B1 (Cat# NB 100–908, RRID: AB_526909), and GAPDH (Cat# NB 100–73,063, RRID: AB_ 1,108,728) were purchased from Novus Biologicals Inc. (Littleton, CO). Fluorescent secondary antibodies including Alexa Fluor® 546 goat anti-mouse IgG (Cat# A-11003, RRID: AB_141370) for ABCA1, Alexa Fluor® 488 donkey anti-rabbit (Cat# A-21206, RRID: AB_1535792) for ABCG1, and Alexa Fluor® 633 donkey anti-goat IgG (Cat# A-21082, RRID: AB_141493) for SR-BI, were purchased from Invitrogen (Carlsbad, CA). Antibodies against phospho-Akt and Akt (Cat# 8200, RRID: AB_ 1,658,157) were purchased from Cell Signaling Technology Inc. (Beverly, MA). Wortmannin were purchased from Cell Signaling Technology Inc. Human ApoA-I, Insulin were purchased from Sigma-Aldrich (St. Louis, MO). Human HDL2 was purchased from Cell Biolabs, Inc. (San Diego, CA). HDL2 subfractions were isolated by sequential density gradient ultracentrifugation from healthy human plasma. The traditional ultracentrifugation method has proved the reliable and the simple available procedure for HDL2 isolation. MCP-1 were purchased from R&D Systems (Minneapolis, MN).
4
Fluorescent Particle Labeling Protocol
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Alexa Fluor 594-conjugated BioParticles® synthesized from killed Escherichia coli (K-12 strain), Staphylococcus aureus (Wood strain without protein A), and Zymosan A (Saccharomyces cerevisiae) were from ThermoFisher Scientific (Waltham, MA). Silica beads were from Kisker Biotech (Moffat Beach, Queensland) and labeled with DQ Green BSA (ThermoScientific) as described [30 ]. Unlabeled oxLDL (lot number 910G18A), along with 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanine perchlorate (DiI)-labeled acLDL (fl-acLDL; J65597; lot numbers 920C18A, 902F18A) and oxLDL (fl-oxLDL; J64164; lot numbers 920C18A, 902F18A) were from Alfa Aesar (Tewksbury, MA). Inhibitors of ROCK1/2 (Y-27632), FAK (PF-573228), and TRPV4 (HC-067047 and GSK2193874) were from Sigma-Aldrich (St. Louis, MO), and used at the indicated concentrations. Primary antibodies were against murine CD36 (monoclonal Armenian hamster; BioLegend; San Diego, CA), SRb1 (polyclonal rabbit; Novus biologicals; Littleton CO), and LOX-1 (polyclonal rabbit; abcam; Cambridge, MA). Dihydroethidium (DHE) was from ThermoFisher Scientific (Waltham, MA).
5
Histological and Immunohistochemical Analysis of Liver and Skin Tissues
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Liver and skin tissue were fixed in 10% buffered formaldehyde and embedded in paraffin. For histological observation, the sections (4 µm thickness) were deparaffinized in xylene and rehydrated in alcohol gradients and then stained with hematoxylin and eosin (H&E). For immunohistochemistry, the sections were dewaxed and rehydrated with the same method for histology. After dewaxing, the slides were incubated overnight at 4℃ with following antibodies: 4-HNE (Millipore Corporation, Billerica, MA, USA), SR-B1 (Novus Biologicals, Inc.; Littleton, CO) and ABCA1 (Abcam, Cambridge, MA). Then the slides were washed three times with phosphate-buffered saline (PBS), and endogenous peroxidase was blocked with 3% H2O2 in absolute methanol for 30 minutes at room temperature (RT). Next, the slides were incubated with EnVision+ System-HRP (DAKO, Glostrup, Denmark) for 45 minutes at RT. Finally, the reaction products were stained with diaminobenzidine (DAB), counterstained with Mayer's hematoxylin, and mounted with Eukitt mounting medium after drying. Images were acquired and analyzed with Axio Vision Release 4.6.3 software.
6
Lipid Metabolism Regulatory Compounds
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Evodiamine (#E3531), phorbol 12-myristate 13-acetate (PMA) (#P1585), water soluble cholesterol (#C4951), ApoA1 (#73366), cycloheximide (#C7698), T0901317 (#T2320), and digitonin (#D141) were purchased from Sigma-Aldrich (Vienna, Austria), and pioglitazone (#M35102242) was obtained from Molekula (Munich, Germany). [3H]-cholesterol (#NET139001MC; 1 mCi, 37 MBq) was provided by Perkin Elmer Life Sciences (Vienna, Austria). The tested compounds were dissolved in DMSO, aliquoted and stored at −20 °C until use. An equal amount of DMSO was always tested in each condition in all experiments to assure that the solvent vehicle does not influence the results itself. Primary antibodies against ABCA1 (#NB400-105), ABCG1 (#NB400-132), ABCA12 (NB100-93466) and SR-B1 (#NB400-104) were obtained from Novus Biologicals (Vienna, Austria). The anti-actin antibody (#8691002) was acquired from MP biologicals (Illkirch, France). Primary antibodies against TRFC (#12113) and LDLR (#SC-18823) were bought from Cell Signaling (Austria) and Santa Cruz (Austria), respectively. HRP-linked anti-rabbit IgG secondary antibody (#7074S) was purchased from New England Biolabs (UK), and horseradish peroxidase conjugated goat anti-mouse secondary antibody (#12-349) from Upstate (Millipore, Vienna, Austria). All antibodies were used in a dilution of 1:500.
7
Immunoblotting Protocol for Protein Analysis
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Unless indicated otherwise, all materials were from Sigma-Aldrich (St. Louis, MO). Geranylgeraniol was a generous gift of Prof. Ewa Sviezewszka (Polish Academy of Science, Warsaw, Poland). For immunoblotting, antibodies against the following proteins were used: P-AMPKa and AMPKa (Cell Signalling Technology, Boston, MA), PP2A (catalytic sub-unit), RhoA, LRP1, and SREBP-1 (Santa Cruz Biotechnology, Santa Cruz, CA), LDLr (ab30532 and SREBP-2 (Abcam, Cambridge, United Kingdom), P-HMGR (Millipore, Temecula, CA), HMGR (Upstate, Lake Placid, NY), SRB-1 (Novus Biological, Littleton, CO). a-Tubulin (Sigma-Aldrich) or caveolin (Santa Cruz, CA) were used as loading controls . HRP-conjugated IgG produced in mouse or in rabbit used as secondary antibodies were obtained from Biorad Laboratories (Milan, Italy).
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