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3 protocols using lipopolysaccharides lps l4391 1mg

1

Antibody Panel for Inflammatory Signaling

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Antibodies against Flag (A8592, 1:1000) and β-actin (A1978, 1:1000) were purchased from Sigma. Antibodies against HA (12013819001, 1:1000) and Myc (11814150001, 1:1000) were purchased from Roche Applied Science. Antibodies against IKK (#05-535, 1:1000) were purchased from Millipore. An antibody against NLRP11 (ab103333, 1:1000) were purchased from Abcam. Antibodies against TRAF6 (sc-8409, 1:500) and RNF19A (sc-55808, 1:500) were purchased from Santa Cruz Biotechnology. Antibodies against the following proteins were purchased from Cell Signalling Tchnology: p-IKKα/β (#2697, 1:1000), p-IκBα (#9246, 1:1000), IκBα (#4814, 1:1000), p-p38 (#9211, 1:1000), p38 (#9212, 1:1000), p-JNK (#9251, 1:1000), JNK (#9252, 1:1000), p-ERK (#9101, 1:1000), ERK (#9102, 1:1000). Recombinant human TNF-α (10 ng/ml) and IL-1β (10 ng/ml) were purchased from Pepro Tech. Lipopolysaccharides (LPS) (L4391-1MG, 100 ng/ml) was purchased from Sigma. Pam3CSK4 (100 ng/ml), poly(I:C) (10 μg/ml) and HKST (106 cells per ml) were purchased from InvivoGen.
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2

Immunoblotting Antibodies and Reagents

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Horseradish peroxidase (HRP)-anti-Flag (M2) (A8592) and anti-β-actin (A1978) were purchased from Sigma; HRP-anti-hemagglutinin (12013819001), anti-c-Myc-HRP (11814150001) were purchased from Roche Applied Science; anti-phospho-IKKα/β (No.2697S), anti-phospho-JNK (No.9251), anti-JNK (No.9252), anti-phospho-ERK (No.9101), anti-ERK (No.9102), anti-phospho-p38 (No.9211), anti-p38 (No.9212), anti-p65 (No.6956), anti-IκBα (No.4814) and anti-phospho-IkBα (Ser32/36) (No. 9246) were from Cell Signaling Technology; anti-NEMO (sc-8300), anti-USP18(sc-98431) were purchased from Santa Cruz Biotechnology; anti-IKKβ were purchased from Millipore; CF568 Goat anti-mouse IgG (H+L) (20101-1) were purchased from Biotium; anti-linear polyubiquitin-specific monoclonal antibody (AB130) were purchased from LifeSensors.
Recombinant human TNF-α was purchased from PeproTech; Lipopolysaccharides (LPS) (L4391-1 MG) were purchased from Sigma. Pam3CSK4 and CL097 were purchased from InvivoGen.
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3

Antibody Characterization and Analysis

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The antibodies used in this study were as follows: HRP-anti-hemagglutinin (12013819001) and anti-c-Myc-HRP (11814150001) (Roche Applied Science); HRP-anti-Flag (M2) (A8592) and anti-β-actin (A1978) (Sigma-Aldrich, St. Louis, MO); anti-phospho-IKKα/β (No. 2697s), anti-phospho-JNK (No.9251), anti-JNK (No. 9252) anti-phospho-ERK (No. 9101), anti-ERK (No. 9102), anti-phospho-ERK (No. 9101), anti-ERK (No. 9102), anti-phospho-p38 (No. 9211), anti-p38 (No. 9212), anti-p65/RelA (No. 6956), anti-IκBα (No. 4814) and anti-phospho-IkBα (Ser32/36) (No. 9246) (Cell Signaling Technology, Danvers, MA)); anti-IKK (Merck Millipore, Billerica, MA); Anti-LRRC25 (ab84954) (Abcam, Cambridge, U.K.); Mouse mAb HA tag DyLight 650(ab117515) (Abcam, Cambridge, U.K.). Recombinant human TNF-α was purchased from PeproTech; Lipopolysaccharides (LPS) (L4391-1 MG) and Bafilomycin A1 were purchased from Sigma-Aldrich (St. Louis, MO). Trizol was purchased from Invitrogen and Transcript First Strand cDNA Synthesis Kit from Roche.
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