Total proteins in cell lysate samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to nitrocellulose membrane26 (link). Blotting of the membrane with antibodies against RIG-I (Santa Cruz Biotechnology, Inc., Dallas, TX) and tubulin (Sigma-Aldrich Corp, St. Louis, MO) was conducted. Horseradish peroxidase-conjugated secondary antibodies (Rockland Immunochemicals Inc.) and chemiluminescence substrate were used to reveal specific reactions by the primary antibodies. Chemi-Doc Imaging System (Bio-Rad, Hercules, CA) was used to capture the luminescence signal.
Rig 1
Manufactured by Santa Cruz Biotechnology
RIG-I is a protein involved in the innate immune response. It functions as a cytosolic pattern recognition receptor that detects the presence of viral RNA in the cell, triggering antiviral signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Merck Group (2 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Rockland Immunochemicals (1 mentions)
Phospho tbk1 ser172, by Cell Signaling Technology (1 mentions)
Mab374, by Merck Group (1 mentions)
Ab108341, by Abcam (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
Dapi solution, by Thermo Fisher Scientific (1 mentions)
Tom20, by Santa Cruz Biotechnology (1 mentions)
β actin, by Bioss Antibodies (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Vdac1, by Santa Cruz Biotechnology (1 mentions)
Ecsit, by Abcam (1 mentions)
Phosphor irf3, by Cell Signaling Technology (1 mentions)
Hdac1, by Santa Cruz Biotechnology (1 mentions)
5 protocols using rig 1
1
Immunoblotting Analysis of RIG-I Protein
2
Protein Extraction and Western Blotting
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Protein extraction from cells using RIPA buffer (human cell lines) or hypotonic lysis buffer (RM1) and Western blotting was done essentially as described previously (39 (link)). Primary antibodies used in human Western blotting were: TBK1 (Cell Signaling Technology 3013; RRID: AB_2199749); phospho-Ser172-TBK1 (Cell Signaling Technology D52C2; RRID: AB_10693472); RIG-I (Santa Cruz Biotechnology SC-376845; RRID: AB_2732794); and GAPDH (Millipore MAB374; RRID: AB_347661). Primary antibodies used in murine Western blotting were: AR (Abcam ab108341; RRID: AB_10865716); and GAPDH XP (Cell Signaling Technology D16h11; RRID: AB_10622025). Horseradish peroxidase–conjugated anti-rabbit and anti-mouse IgG secondary antibodies (Dako) were used and immunoreactive bands visualized using Clarity Western ECL Substrate (Bio-Rad).
3
Profiling Pattern Recognition Receptors in APCs
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To analyze the activation and the expression of PRR (TLR3, MDA5 and RIG-I) within blood DC and monocytes, PBMC were incubated for 30 min on ice with isotype-matched control antibodies for lymphocytes and granulocytes (lin-1), DC (HLA-DR, CD11c, CD123 and CD86) and monocytes (CD14). Monocytes were defined as CD14+ cells. cDC and pDC subsets were respectively defined by the Lineage1− CD14− HLA-DR+ CD123+ and Lineage1− CD14−CD11c+ HLA-DR+ phenotypes as illustrated in the Additional file 1 . Cell activation in APC was analyzed by measurement of the median of fluorescence (MFI) for HLA-DR and CD86 (BD-Biosciences). Moreover, the expression of TLR3, RIGI and MDA5 (Santa-Cruz Biotechnology) was estimated by indirect labeling after cell permeabilization. A corresponding isotype control was included to define the background level and the results were expressed after subtraction of the value obtained with the isotype control.
4
Protein Quantification and Visualization
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Cell lysates were quantified by Bradford assay. According to the detected protein size, SDS-PAGE gels at various percentages (8–13.5%) were used to separate proteins. For immunoblotting and immunofluorescence, antibodies against β-actin (Bioss, cat. #BS-0061R, Woburn, MA, USA), GAPDH (Millipore; cat. #MAB374, Burlington, MA, USA or Santa Cruz; cat. #sc-47724, Dallas, TX, USA), VDAC1 (Santa Cruz, cat. #sc-8828), MT-CO2 (Life Technologies, cat. #A6404, Carlsbad, CA, USA), p65 (Santa Cruz, cat. #sc-372 or sc-8008), phosphor-IRF3 (Cell Signaling, cat. #4947S, Danvers, MA, USA), ECSIT (Abcam, cat. #ab21288), MAVS (Santa Cruz, cat. #sc-166583), RIG-I (Santa Cruz, cat. #sc-376845), NDV HN (Santa Cruz, cat. #sc-53562), influenza A virus NP (Abcam, cat. #ab128193), TOM20 (Santa cruz, cat. #sc-17764) and HDAC1 (Santa Cruz, cat. #sc-6298) were used. For immunofluorescence staining, cells were seeded on cover glass that had been coated with poly-D-lysine. Then, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 1% BSA. An Alexa Fluor 488-conjugated secondary antibody (Invitrogen, cat. #A11017) was used in this study. To stain mitochondria, cells were incubated with 100–400 nM MitoTracker Deep Red (Invitrogen, cat. #M22426) in serum-free DMEM for 30 min. DAPI solution (Invitrogen, cat. #R37606) was used to visualize nuclei.
5
Protein Detection Methodology and Analysis
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Proteins were detected by designated antibodies as following: MDA5 (D74E4, Rabbit mAb, Cell Signaling #5321S, 1:1,000), MDA5 (Santa Cruz, sc-48031, 1:1,000), RIG-I rabbit Ab (Cell Signaling, #4520S, 1:1,000), RIG-I (Santa Cruz, sc-48929, 1:1,000), B7-2 (BU63, sc-19617, Santa Cruz Biotechnology, 1:1,000), B7.2 (Santa Cruz, sc-28347, 1:1,000), Claudin-1 mAb (ZYMED, #37-4900, 1:2,000). HCV E2 was detected by CBH5 or CBH17 antibodies (kind gifts from Prof. Steven Foung in Stanford University, Palo Alto, CA, USA, 1:1,000). The phosphorylation of PLCγ2 (Tyr1217: Cell Signaling cat#: 3871 T, 1:2,000) and PKCα/β II (Thr638/641: Cell Signaling cat#: 9375 T, 1:2,000) were analysed using antibodies directed against their respective phosphorylated forms. Total protein levels were determined using antibodies not specific for the phosphorylated forms of these proteins. Uncropped scans of western blots are provided in Supplementary Fig. 10 .
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