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2 protocols using anti phospho p p38

1

Molecular Mechanisms in Inflammatory Signaling

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The antibodies included anti-S100A9 (Cat no. ab92507; Abcam), anti-TLR4 (Cat no. sc-293072; Santa Cruz Biotechnology), anti-RAGE (Cat no. sc-80653; Santa Cruz Biotechnology), anti-p38 (Cat no. 9212; Cell Signaling Technology), anti-p65 (Cat no. 3034; Cell Signaling Technology), anti-ERK1/2 (Cat no. 4695; Cell Signaling Technology), anti-JNK (Cat no. 9253; Cell Signaling Technology), anti-AKT (Cat no. 8596; Cell Signaling Technology), anti-phospho(p)-p38 (Cat no. 4511; Cell Signaling Technology), anti-p-p65 (Cat no. 3033; Cell Signaling Technology), anti-p-ERK1/2 (Cat no. 3510; Cell Signaling Technology), anti-p-JNK (Cat no. 4668; Cell Signaling Technology), anti-p-AKT (Cat no. 9271; Cell Signaling Technology), anti-CD8 (Cat no. 340046, BD), anti-HLA-DR (Cat no. 4310370, eBioscience), anti-CD33 (Cat no. 4296343, eBioscience) and CD11b (Cat no. 4291932, eBioscience), and horseradish peroxidase-conjugated anti-mouse, anti-rabbit IgG antibodies. The inhibitors contained TAK-242 (MedChemExpress, New Jersey), FPS-ZM1 (MedChemExpress, New Jersey), SB203580 (Beyotime) and BAY 11-7082 (Beyotime). The preparation of the recombination GST-S100A9 protein, as well as its control protein GST, have previously been described (21 (link)).
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2

Isolation and Culture of Splenic Macrophages

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Highly purified LPS from Escherichia coli O111:B4 and loxoribine (Lxrb) were from Invivogen (San Diego, CA). The following Abs were used: anti-phospho (p)-p38 and anti-p38, anti-p-IRAK1, anti-p-p65 and anti-p65 (Cell Signaling Technology, Danvers, MA), anti-IRAK1, anti-IRAK-M, anti-Tollip, anti-pERK1/2 and anti-ERK1/2, anti-pJNK1/2 and anti-JNK1/2, and anti-tubulin (Santa Cruz Biotechnology, Dallas, Texas). Splenic MΦs were obtained by differentiation of splenocytes with CSF-1-containing L929-conditioned medium [78 (link)]. This procedure led to a significant enrichment of CD11b+F4/80+ splenic MΦs (Fig. S3) [78 (link)]. Splenocytes were plated into 10-cm tissue culture dishes and cultured for 7 days in RPMI medium supplemented with 2 mM L-glutamine, 100 u/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 10% FBS (Atlanta Biologicals, Flowery Branch, GA), 5 × 10−5 M β-mercaptoethanol (complete RPMI), and 25% L929-conditioned medium. Following trypsin treatment (0.5%), cells were resuspended in complete RPMI and used for experiments.
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