The IL-33-pGL3 reporters (−1050 to +49, −662 to +49, −520 to +49, −460 to +49, −300 to +49, −160 to +49) were generated by cloning the PCR products from THP-1 cell genomic DNA into the pGL3-basic vector (Promega). The IRF7 binding site mutants (Mut.A, Mut.B and Mut.AB) were generated by GeneArt® Site-Directed Mutagenesis System from Life Technologies. Human IRF7 sequence was amplified by PCR with total cDNA from THP-1 cells, which was cloned into pcDNA3.0-HA vector. All primers used were shown in the Supporting Information Table 1 . Recombinant human SAA was obtained from PeproTech (Rocky Hill, NJ). cycloheximide (CHX) and actinomycin D (ActD) was purchased from Sigma-Aldrich (St Louis, MO). The TRAF6 plasmid, Pam3CSK4, polyI: C, TLR2 neutralizing antibody and its isotype control IgA were obtained from InVivoGen (San Diego, CA). Anti-IL-33 (ab118503) and anti-IRF7 were obtained from Abcam (Cambridge, MA). Anti-pSer/Thr was obstained from BD Biosciences (San Jose, CA). Antibodies for caspase-3, β-actin, HDAC1, HRP-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG were obtained from Cell Signaling Technology (Danvers, MA).
Anti pser thr
Manufactured by BD
Anti-pSer/Thr is a laboratory reagent used for the detection and quantification of phosphorylated serine and threonine residues in proteins. It is a tool used in biochemical and cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Geneart site directed mutagenesis system, by Thermo Fisher Scientific (1 mentions)
Anti irf7, by Abcam (1 mentions)
Recombinant human saa, by Thermo Fisher Scientific (1 mentions)
Actinomycin d actd, by Merck Group (1 mentions)
Hrp conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)
Hdac1, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Ab17057, by Abcam (1 mentions)
Plk1 pt210, by Abcam (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
2 protocols using anti pser thr
1
Cloning and Mutagenesis of IL-33 Reporters
2
Immunoprecipitation and Western Blotting Protocol
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Cells were lysed using lysis buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.5% NP‐40 and cocktail protease inhibitors). The cell lysate was incubated with indicated antibodies at 4°C overnight, followed by the addition of protein G‐coated agarose beads (Invitrogen). After incubation at 4°C for 4 h, beads were washed three times with the lysis buffer. The precipitated components were eluted by boiling the beads in Sodium dodecyl sulfate loading buffer. Western blotting was performed as previously described. Primary antibodies were listed as: METTL16 (Sigama, HPA020352), METTL16 (abconal, A15894), Soga1 (Santa, sc‐514,885), GAPDH (abconal, AC001), anti‐p‐Ser/Thr (BD Biosciences, 612,549), PLK1 (abcam, ab17057), PLK1‐pT210 (abcam, ab155095), Flag (CST, 14793), Flag (Sigma, F3165), HA (abconal, AE008), HA (abconal, AE036).
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