Journal: The Journal of Biological Chemistry

Article Title: Phosphodiesterase 10A Is Tethered to a Synaptic Signaling Complex in Striatum *

doi: 10.1074/jbc.M114.595769

Figure Lengend Snippet: Characterization of pPDE10 antibody. Heterologously expressed PDE10A2 was incubated with ATP (0.5 mm) and MgCl2 (3 mm) in the absence or presence of PKA (800 units) for 30 min at 37 °C. An aliquot of the phosphorylated sample was saved for Western blot before purification of pPDE10 using the Pierce phosphoprotein enrichment kit. Samples were analyzed using PDE10 and pPDE10 antibodies. Graphs depict densitometric analysis of PDE10 phosphorylation. Shown is the ratio of phospho-PDE10 to PDE10 signal before and after purification of the phosphorylated protein.

Article Snippet: The cell extract was diluted with PKA buffer (0.5 m m ATP, 3 m m MgCl 2 , 0.5 mg/ml of BSA, 2 m m dl -dithiothreitol, 50 m m triethanolamine/hydrochloride, pH 7.4, final concentrations) and incubated with or without 0.5 μg (800 units) of PKA (Jena Biosciences, PR-318) for 30 min at 37 °C.

Techniques: Incubation, Western Blot, Purification

Journal: The Journal of Biological Chemistry

Article Title: Phosphodiesterase 10A Is Tethered to a Synaptic Signaling Complex in Striatum *

doi: 10.1074/jbc.M114.595769

Figure Lengend Snippet: PDE10A interacts with PKA. PDE10A was precipitated from lysates of mouse striatum using specific antibodies coupled to Dynabeads. A, the precipitates were incubated with or without ATP (0.5 mm), MgCl2 (3 mm), and cAMP (10 μm) and PDE10A2 phosphorylation was detected using pPDE10A antibodies. Shown are representative blots and mean ± S.E. of pPDE10A/PDE10A ratio after densitometric analysis; n = 3; *, p < 0.005, Student's t test. B, immunoprecipitation (IP) of PDE10 was performed in the absence or presence of the PDE10A antigen used for antibody generation and PKA RIIα was detected by Western blot. Larger sections of Western blots are shown in supplemental Fig. S4.

Article Snippet: The cell extract was diluted with PKA buffer (0.5 m m ATP, 3 m m MgCl 2 , 0.5 mg/ml of BSA, 2 m m dl -dithiothreitol, 50 m m triethanolamine/hydrochloride, pH 7.4, final concentrations) and incubated with or without 0.5 μg (800 units) of PKA (Jena Biosciences, PR-318) for 30 min at 37 °C.

Techniques: Incubation, Immunoprecipitation, Western Blot