Cell Signaling Technology
Rabbit anti–phospho-IRF3 antibody
4947S
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Protein extract from cells was prepared in radioimmunoprecipitation assay lysis buffer (sc-24948; Santa Cruz Biotechnology) supplied with PMSF, protease inhibitor mixture, sodium orthovanadate, or RNase inhibitor. Lysates were quantified using a Bio-Rad protein assay kit. Forty micrograms of proteins were subjected to SDS-PAGE and transferred onto nitrocellulose membranes and blocked with 5% non-fat milk in TBS-T (20 mMTris, 500 mMNaCl, and 0.1% Tween 20) at room temperature for 1 h with rocking. The membranes were probed with primary Antibody (1/1000 dilution) overnight at 4°C. After washing with TBS-T, the membranes were incubated with HRP-conjugated secondary Antibodies (Thermo Scientific, Rockford, IL) in 5% non-fat milk/TBS-T at room temperature for 1 h. The protein–Antibody complex was detected by ECL substrate (cat # 32106; Thermo Scientific).
Western Blot
Mouse MEFs
4 degrees Celsius overnight
5% milk
Goat anti-rabbit secondary antibody
Room temperature 1 hour
ECL
RIG-I and MDA5’s downstream biochemistry modifications on IRF3 and IkB-α were monitored by Western blot with specific antibodies against the phosphorylated proteins after SeV infection or poly I:C transfected into the iKO-MEFs.
See Supplementary Figure 1D at http://www.jimmunol.org/content/suppl/2014/08/28/jimmunol.1401136.DCSupplemental/JI_1401136_Supplemental_Figures_1.pdf
Works well
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Works as expected