Western blots were conducted as previously described (Markovics 2005 (link)) with the following antibodies: Total STAT1 (Cell Signal #9172), Phospho-STAT1 Ser727 (Cell Signal #9177), and Total STAT2 (Cell Signal #4597) and were prepared in accordance with the manufacturer’s instructions. SV40 T Antigen mouse monoclonal antibodies have been described previously: PAb416, PAb419 (Harlow 1981 (link)), and PAb901 (Fu 1996 (link)). Additional mouse monoclonal antibodies against JCV T antigens (PAb962 and AB2003) were previously described (Bollag 2000 (link), Munoz-Marmol 2004 (link)). GAPDH mouse monoclonal was used as the loading control (US Biologicals #G8140-11). Peroxidase-conjugated goat anti-mouse (A2554), and goat anti-rabbit (A0545) from Sigma-Aldrich were used as secondary antibodies. The Luminata Western HRP substrate (Millipore #WBLUF0100) was used in accordance with manufacturer’s instructions. A mixture of monoclonal antibodies 416/419/962/2003 (1:1000/1:500/1:1000/1:1000) were used for the detection of T antigen signal in Figures 2C , 3A , 4B , and 7B . Additionally, SV40-C257 detection used monoclonal Ab901 (1:500) Figure 5D , and in Figure 5B a mixture of 416/419 (1:500/1:250) was used.
Total stat2
Manufactured by Cell Signaling Technology
Sourced in United States
Total Stat2 is a quantitative immunoassay kit designed to measure the total levels of Stat2 protein in cell and tissue lysates. The kit utilizes a proprietary antibody-based detection method to provide accurate and reproducible Stat2 quantification.
Automatically generated - may contain errors
Lab products found in correlation
Total stat1, by Cell Signaling Technology (4 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
L nmma, by Merck Group (2 mentions)
Pe anti mouse h 2kb h 2db, by BioLegend (2 mentions)
Ptyr701 stat1, by Cell Signaling Technology (2 mentions)
Total iκbα, by Cell Signaling Technology (2 mentions)
Pe anti mouse ifnγr β chain, by BioLegend (2 mentions)
Total p65 pser32 iκbα, by Cell Signaling Technology (2 mentions)
Ptyr690 stat2, by Abcam (2 mentions)
Lamin b, by Abcam (2 mentions)
Recombinant mouse ifnα, by R&D Systems (2 mentions)
P p65 ser536, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Ifn γ, by R&D Systems (2 mentions)
Ifn β, by R&D Systems (2 mentions)
Luminata western hrp substrate, by Merck Group (1 mentions)
Phospho stat1 ser727, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated goat anti mouse a2554, by Merck Group (1 mentions)
Recombinant human ifn β, by PBL Assay Science (1 mentions)
4 protocols using total stat2
1
Western Blot Analysis of STAT and T Antigens
2
STAT1/2 Activation and Antiviral Effectors in ZIKV-Infected Cells
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STAT1 and STAT2 signaling was studied in A549 cells as previously described [61 (link)]. Briefly, A549 cells were infected with the indicated ZIKV strain at an MOI of 0.1 and 1 (based on Vero cell titration). At 48hpi, cells were pulse treated with 1000 IU/mL of recombinant human IFNβ (PBL Assay Science) for 30 minutes and whole-cell lysates were collected in modified radioimmunoprecipitation assay buffer supplemented with Halt Protease Inhibitor Cocktail (ThermoFisher) and phosphatase inhibitor cocktail II (Calbiochem). Western blot analysis was performed to detect STAT1 phosphotyrosine residue 701 (Cell Signaling), total STAT1 (Cell Signaling), STAT2 phosphotyrosine residue 689 (Upstate, EMD Milipore), total STAT2 (Cell Signaling), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling). Protein expression levels were quantified using Image Lab software. For analysis of antiviral effector proteins within human moDCs, 4e5 cells were used per condition and protein lysates were collected as described for A549 cells. The following antibodies were obtained from Cell Signaling: RIG-I, MDA5, LGP2, STAT1, STAT2, IFIT1, viperin, and GAPDH. The IFIT3 antibody was kindly provided by Dr. G. Sen.
3
Flow Cytometry and Western Blot Analysis of Interferon Signaling
4
Flow Cytometry and Western Blotting of IFN Signaling
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The following antibodies were used in flow cytometry: PE anti-mouse H-2Kb/H-2Db, PE anti-mouse IFNγR β chain (Biolegend, San Diego, CA, USA) and PE anti-mouse CD119 (IFNγ Receptor 1) (eBioscience, San Diego, CA, USA). Antibodies used in western blotting analysis were iNOS, pTyr701-Stat1, total Stat1, total Stat2, pSer536-p65, total p65 pSer32-IκBα, total IκBα, β-actin, GAPDH (Cell Signaling Technology, Danvers, MA, USA); pTyr690-Stat2 (Abcam, Cambridge, MA, USA) and LaminB (Epitomics, Burlingame, CA, USA). Recombinant mouse IFNα, IFNβ, IFNγ and TNFα were from R&D Systems (Minneapolis, MN, USA). l -NMMA was from Sigma-Aldrich (St Louis, MO, USA).
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