Journal: Journal of Cellular and Molecular Medicine

Article Title: S100B induces tau protein hyperphosphorylation via Dickopff-1 up-regulation and disrupts the Wnt pathway in human neural stem cells

doi: 10.1111/j.1582-4934.2008.00159.x

Figure Lengend Snippet: S100B induces AP-1/DNA complex activation through RAGE interaction. ( A ) AP-1/DNA complex activation was detected in NSCs at various time point (30 min. to 6 hrs) following the exposure to 5 μM of S100B. The time course of AP-1/DNA binding activity was evaluated by EMSA (upper panel) and densitometric analysis of corresponding bands (lower panel). ( B ) AP-1/DNA binding activity induced by 5 μM of S100B in NSCs in the presence or absence of two different concentrations of RAGE blocking antibody or unrelated blocking antibody (1:1000 or 1:10,000). Statistics show that S100B significantly enhanced the activation of AP-1/DNA complex and that the specific RAGE blocking antibody significantly blunted this effect, while the unrelated antibody failed to influence it. The AP-1/DNA binding activity was measured 2 hrs after S100B exposure by EMSA analysis (upper panel). The lower panel shows densitometric analysis of corresponding bands. ( C ) AP-1 antibodies (anti-Fra-1, anti-c-Fos, anti-c-Jun and anti-Jun-D) were preincubated with nuclear extracts from NSCs exposed to 5 μM of S100B. Results of supershift analysis (upper panel) demonstrate the effect of the antibodies on the changes in the relative mobility of AP-1 species 2 hrs following NSC stimulation with S100B. Densitometric analysis of corresponding bands is reported in the lower panel. ( D ) NSCs were challenged with increasing concentrations of S100B (0.05–5 μM) and lysed 6 hrs later. c-Jun mRNA expression was evaluated by RT-PCR (upper panel) and densitometric analysis of corresponding bands (lower panel). Statistics demonstrate significant and concentration-dependent effect of S100B on c-Jun mRNA expression. The figure also shows two different dilutions of RAGE blocking antibody (1:1000 or 1:10,000) were able to revert the effect of the highest concentration of S100B, whereas the same dilutions of an unrelated blocking antibody were ineffective. Results are the mean ±S.E.M. of three independent experiments.*** P < 0.001, ** P < 0.01, and * P < 0.05 versus unchallenged cells (ctrl);°°° P < 0.001 and °° P < 0.01 versus 5 μM S100B challenged cells.

Article Snippet: Samples were incubated with 10000 cpm of the 32 P-labelled AP-1 (5′-CGCTTGATGAGTCAGCCGGAA-3′) oligonucleotide (Promega Corp., Madison, WI, USA).

Techniques: Activation Assay, Binding Assay, Activity Assay, Blocking Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay

Journal:

Article Title: Molecular and pharmacological characterization of a functional tachykinin NK 3 receptor cloned from the rabbit iris sphincter muscle

doi: 10.1038/sj.bjp.0702854

Figure Lengend Snippet: (a) NK3 receptor mRNA distribution in a whole rabbit eye section (×5.8) labelled with 5′-CTTCTGGCCATTGCACATAGCAAAGAGTACGTCCAGGC antisense probe, showing discrete hybridization to iris muscle and associated ciliary processes; (b) higher magnification (×10) showing discrete localization of NK3 receptor mRNA to ciliary processes, and (c) sections (×5.8) exposed to 5′-GCCTGGACGTACTCTTTGCTATGTGCAATGGCCAGAAG sense probe showing minimal hybridization throughout the eye.

Article Snippet: In situ hybridization histochemistry Avoiding homology to other known receptors, especially NK 1 and NK 2 receptors, and using the sequence data generated from the specific NK 3 receptor cDNA obtained from the rabbit iris sphincter muscle, specific oligonucleotide probes for in situ hybridization histochemistry (ISHH) were designed and obtained from Genosys (Cambridge, U.K.):- antisense, 5′-CTTCTGGCCATTGCACATAGCAAAGAGTACGTCCA-GGC; sense, 5′-GCCTGGACGTACTCTTTGCTATGTGCAATGGCCAGAAG.

Techniques: Hybridization