Journal: The Journal of Neuroscience

Article Title: Cocaine- and Amphetamine-Regulated Transcript: Stimulation of Expression in Rat Vagal Afferent Neurons by Cholecystokinin and Suppression by Ghrelin

doi: 10.1523/JNEUROSCI.5508-06.2007

Figure Lengend Snippet: CCK induces CREB phosphorylation through activation of PKC in vagal afferent neurons. Vagal afferent neurons cultured for >48 h and stained with antibody to phosphoCREB (A, C, E, G, I, K) or CREB (M, O) and DAPI (B, D, F, H, J, L, N, P), in the respective fields, are shown. A, B, Nuclear phosphoCREB staining was undetectable after transfer to serum-free medium for 2 h. C–F, Nuclear phosphoCREB was readily detectable after treatment with 10 nm CCK (C, D) and 100 nm PMA (E, F) for 30 min. G, H, The effect of CCK was inhibited by pretreatment with 1 μm Ro-32-0432. I, J, Nuclear phosphoCREB was undetectable after treatment with ghrelin (10 nm). K, L, Importantly, ghrelin (10 nm) in the presence of 10 nm CCK led to exclusion of phosphoCREB from the nucleus. M–P, Total CREB (M, N) was unchanged by CCK and showed a cytoplasmic localization in the presence of ghrelin plus CCK (O, P). A–L, Arrows identify a representative neuron in each pair of images; filled arrows show phosphoCREB-positive neurons, and open arrows show phosphoCREB-negative neurons. M–O, Arrows indicate a representative neuron exhibiting nuclear localization of total CREB (M, N) and a representative neuron exhibiting cytosolic localization of total CREB (M, O). Representative images from six independent experiments are shown. Scale bar, 50 μm.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibody to CART (Phoenix Laboratories, San Antonio, TX); affinity-purified rabbit polyclonal antibody to phosphoCREB (Cell Signaling Technology, Beverly, MA); affinity-purified goat polyclonal antibody to MCH; rabbit polyclonal antibody to the CCK-1 receptor; rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; Santa Cruz Biotechnology, Calne, Wiltshire, UK); affinity-purified mouse monoclonal antibody to neuron-specific enolase (NSE; Abcam, Cambridge, UK); and affinity-purified mouse anti-Flag monoclonal antibody (Upstate Biotechnology, Hampshire, UK).

Techniques: Activation Assay, Cell Culture, Staining

Journal: bioRxiv

Article Title: Myomegalin regulates Hedgehog pathway by controlling PDE4D at the centrosome

doi: 10.1101/2020.04.24.059923

Figure Lengend Snippet: (A) Diagram showing the proteolytic processing of Gli3 after PKA phosphorylation. (B) 24hr SAG treatment reduced Gli3R levels in wild type cells, but not in Mmg knockout cell clones. (C) Representative images of Gli2 immunostaining after cells are stimulated with SAG. White arrows point to Gli2 at the cilium tips. (D) Quantification of Gli2 levels at the cilium tips. Mmg knockout reduced Gli2 levels at the cilium tips, while Mmg overexpression restored Gli2 intensity. Data are shown as mean ± SD. Statistics: Kruskal–Wallis non-parametric One-Way ANOVA, followed by Dunn’s multiple comparison. ****p<0.0001. A.U.: arbitrary unit. (E-F) Diagram showing PED4D3 specifically controls PKA activities at the centrosome to regulate the Hh signaling transduction. Under normal conditions, Upon Shh stimulation, SMO is translocated and activated in the cilium, which then triggers a signaling cascade that reduces cAMP levels at the cilium base. The subsequent inhibition of PKA allows Gli2 to be translocated and activated in the cilium tips (E). Without myomegalin, PDE4D3 is dislocated from the centrosome and fails to degrade the local cAMP. Thus, PKA levels remain high at the centrosome even after Shh stimulations. Hyperactive PKA suppresses Gli2 activation and promotes Gli3R production. As a result, the Hh pathway cannot be activated (F).

Article Snippet: Primary antibodies used: mouse anti-GAPDH (ab9484, Abcam), rabbit anti-phosphoPKA-T197 (5661S, Cell Siganling), rabbit anti-phosphoCREB-S133 (9198S, Cell Signaling), mouse anti-PKA (610625, BD Biosciences), rabbit anti-Gli1 (V812, Cell Signaling), rabbit anti-Myomegalin (PA5-30324, Invitrogen).

Techniques: Knock-Out, Clone Assay, Immunostaining, Over Expression, Transduction, Inhibition, Activation Assay