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3 protocols using c jun h79

1

Immunoblot Analysis of Signaling Pathways

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Immunoblot analysis was performed after 8 or 12% SDS-PAGE, with overnight incubation with a 1:1,000 dilution of primary antibody and followed by a 1:5,000 dilution of horseradish peroxidase–conjugated anti–rabbit, anti–mouse, or anti–goat antibody (GeneTex). Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The homemade antibody against IL-17RB (A68) was used. Antibodies against CCL20 (67310), CXCL1 (20326), and IL-8 (6217) were purchased from R&D Systems. Antibodies against TFF1 (EPR3972), TAK1 (GTX107452), p84 (GTX118740), ATF2-phosphoThr71 (E268), ATF2 (E242), IKKα (GTX27609), IKKβ (GTX105690) and p65 (GTX102090) were purchased from Genetex. Antibodies against p-ERK1/2 (4370), ERK1/2 (4695), p-IKK (2687), c-JUN-phosphoSer63 (9261), Akt (9272), and p-Akt (4058) were purchased from Cell Signaling Technology. Antibodies against TRAF6 (Sc-8409), ACT1 (H300), AML1α (N20), and c-JUN (H79) were purchased from Santa Cruz Biotechnology. Protein concentration was determined by the Bradford assay (Bio-Rad Laboratories) before loading and verified by α-tubulin or GAPDH level using a 1:100,000 dilution of anti-tubulin antibody (GeneTex). The optical density was determined using the National Institutes of Health ImageJ program.
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2

Antibody Panel for Cell Signaling Analysis

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The antibodies used were as follows: NF-κB p65 (Abcam; cat. no. ab7970), Pol II (Santa Cruz; cat. no. SC-899 and SC-900, mixed in a 1:4 ratio), β-tubulin (Abcam; cat. no.ab6046), SNRP70 (Abcam; cat. no ab51266), c-Fos (H-125, Santa Cruz; cat. no. sc-7202), c-Jun (H-79, Santa Cruz; cat. no. sc-1694), and NF-κB p50 (Abcam; cat. no. ab7971).
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3

Quantification of Protein Expression

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Total protein was isolated using the total protein extraction kit (BestBio Science), and the concentrations were measured using BCA Protein Assay Kit (Beyotime Biotechnology). Proteins were separated on the 10% SDS PAGE gels, and subsequently transferred to polyvinylidene fluoride membranes (PALL corporation). The membranes were probed with primary antibodies Fra-1 (R-20) (1:500 dilution) (Santa Cruz Biotechnology), c-Jun (H-79) (1:500 dilution) (Santa Cruz Biotechnology), or OLFML2A (1:500 dilution) (Abcam plc., UK) made from rabbit, and then probed with donkey anti-rabbit IgG antibody (1:10,000 dilution) (GE Healthcare Company). The protein levels were normalized by probing β -actin antibody (1:10,000 dilution) (Sigma-Aldrich LLC.) and sheep anti-mouse IgG antibody (1:10,000 dilution) (GE Healthcare Company). Immunosignals were imaged using a Tanon 5200 Multi Automatic Chemiluminescence / Fluorescence Image Analysis System (China).
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