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. 2012 Jul 9;198(1):127–141. doi: 10.1083/jcb.201205025

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© 2012 Fontana et al.

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Figure 4. — Molecular analysis of GDNF and Artn promoters. (A) Western blots of IMS32 SC line extracts to detect total and phosphorylated versions of both c-Jun and JNK upon JNK activation (anisomycin, Anm) or JNK inhibition (SP600125) treatment. Actin was used as loading control. (B) Q-PCR from IMS32 SC line samples untreated (white bars) or treated either with anisomycin (black bars) or JNK inhibitor (SP600125; gray bars). Gene values were normalized to the untreated condition (baseline value 1). *, P < 0.05, ANOVA test, n = 3 independent experiments. (C and D) Scheme showing the structure of Mus musculus GDNF (C) and Artn (D) loci. The arrow indicates the transcriptional start site. Exons are shown in black and the UTR regions in gray. The red dot indicates the location of the identified AP-1 site in the promoter in each of these genes. A 5′–3′ species alignment of the AP-1 sequence is shown. The red arrows flanking the AP-1 site indicate the location of the primers used in K. (E, G, and I) Schemes of the pGL3 luciferase reporter constructs into which GDNF (E) or Artn (G) or Jun (I) mouse promoter fragments were cloned. The red dot indicates AP-1–binding sequence. AP-1 sites of GDNF and Artn reporter constructs were mutated (AP-1mut; indicated with a cross). Approximate sizes of the cloned piece of genome, flanking the AP-1, are indicated. (F, H, and J) IMS32 SC cultures were cotransfected with each of these constructs together with ubiquitin-renilla as a control. The graphs show renilla/luciferase ratio of fold induction over pGL3-control (ctrl) transfected cells. Cultures were either untreated or treated with JNK agonist anisomycin. *, P < 0.05, Student’s t test; n = 3 independent experiments. (K) Graph demonstrating phospho-c-Jun in vivo binding to c-Jun, GDNF, and Artn AP-1 containing promoters by chromatin immunoprecipitation using anti–phospho-c-Jun antibody. Location of AP-1 flanking primers used for GDNF and Artn genes is marked in C and D with red arrows. Gapdh gene was used as a control. IMS32 SC line cultures were serum starved overnight and were either untreated or treated with anisomycin or JNK inhibitor for 30 min. The values represent fold enrichment over an isotype matching control immunoglobulin, showing a representative experiment out of three repeats.