Journal: The Journal of Neuroscience

Article Title: The Cytokine Interleukin-6 Is Sufficient But Not Necessary to Mimic the Peripheral Conditioning Lesion Effect on Axonal Growth

doi: 10.1523/JNEUROSCI.0815-06.2006

Figure Lengend Snippet: IL-6 increases in response to either elevated cAMP or a conditioning lesion. A, Dissociated DRG cells were either treated in culture for 18 h with 1 mm db-cAMP or removed from the animal 18 h after a peripheral lesion (CondLesion), and mRNA was isolated and subjected to either microarray analysis or Q-RT-PCR for IL-6. The control was mRNA from untreated, unlesioned animals. Each experiment was performed at least three times. B, Radiolabeled consensus CRE (lanes 1–4) and IL-6 CRE (lanes 5–8) probes were incubated with 25 ng (lanes 2 and 6), 50 ng (lanes 3 and 7), and 100 ng (lanes 4 and 8) of PKA-phosphorylated recombinant CREB. C, Unlabeled, annealed oligomers representing the CRE consensus (CRE wt) and IL-6 CRE (IL-6 wt) sequences were added in 50-fold (lanes 3 and 8), 100-fold (lanes 4 and 9), and 200-fold (lanes 5 and 10) excess over labeled CRE consensus or IL-6 CRE probes to compete for binding of 50 ng of PKA-phosphorylated recombinant CREB (lanes 2–5 and 7–12). A mutated IL-6 CRE sequence (IL-6 mt) was also used to compete for binding of protein at 50-fold (lane 11) and 100-fold (lane 12) excess over labeled probe. D, DRG cells were cultured with 1 mm db-cAMP for the times indicated, after which the cells were lysed and subjected to immunoblotting for IL-6. Recombinant IL-6 (rIL-6) was used as a positive control. E, Hippocampal neurons (HN) or DRG neurons were cultured with (▧) or without (▪) 1 mm db-cAMP for 24 h, after which time the supernatants were collected and subjected to an ELISA for IL-6. Results are expressed as the picomole of IL-6 secreted from 106 cells in that time. Error bars indicate SE of at least three experiments.

Article Snippet: Binding reactions contained 50,000–100,000 cpm of labeled probe, various amounts of phosphorylated recombinant CREB (Active Motif, Carlsbad, CA) [phosphorylated by protein kinase A (PKA) using an in vitro kinase assay (Upstate, Charlottesville, VA)] in binding buffer (20 m m HEPES, pH 7.9, 25 m m KCl, 0.1 m m EDTA, 5% glycerol, 2 m m MgCl 2 , 0.5 m m DTT, 0.025% NP-40, and 0.1 mg/ml BSA) in a total volume of 20 μl, and a 20-fold mass increase poly(dI·dC) over probe.

Techniques: Isolation, Microarray, Reverse Transcription Polymerase Chain Reaction, Control, Incubation, Recombinant, Labeling, Binding Assay, Sequencing, Cell Culture, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Neuroscience

Article Title: The Right Time for a Synapse to Change: Windows and Mechanisms of Multiday Training Trials

doi: 10.1523/JNEUROSCI.1981-25.2026

Figure Lengend Snippet: Phosphorylation of CREB1 and CREB2 after the first stimulus block. A1 , Confocal images of SNs after pCREB1 immunostaining. DAPI was used as a nuclear counterstain. Merged images indicated immunoreactivity to pCREB1 was highest in the nucleus. A2 , Summary data. Different colored circles in each group indicate individual experiments, and the same color in different groups represents one set of experiments. Data in this figure and all other figures are represented as mean ± SEM. ∗ p < 0.05. A3 , No significant (N.S) difference was found in pCREB1 between 5-HT (S) and vehicle (V) groups. B1 , Confocal images of SNs after pCREB2 immunostaining. Immunoreactivity to pCREB2 was also highest in the nucleus. B2 , B3 , Summary data. C , Changes in pCREB1 and pCREB2 at 18 and 24 h. Scale bars, 15 µm.

Article Snippet: The antibody to phosphorylated CREB1 (pCREB1, Genemed Synthesis) utilized the Ser85-phosphorylated CREB1 peptide KKRREILTRRPSYRK epitope ( ).

Techniques: Phospho-proteomics, Blocking Assay, Immunostaining