h89 dihydrochloride  (TargetMol)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A PKA-selective inhibitor captures an open but more ordered conformation of the PKA catalytic subunit

doi: 10.1073/pnas.2536312123

Figure Lengend Snippet: The conformational, dynamic, and kinetic differences between BLU0588 ( A ) and H89 ( B ). Left panels: Comparison of the PKA-C B-factor dynamics when bound to the respective inhibitor. Middle panels: Inhibitor-bound active site interactions and occupation of each PKA-C subsite. Right panels: SPR sensorgrams show the inhibitor’s association and dissociation for BLU0588 and for H89 using a concentration series of the respective inhibitors interacting with captured FSS-tagged PKA-C. Dashed lines are fits to determine the respective rate constants (k ass and k diss ) and the experimental R max for both inhibitors. Representative experiment of n = 2 experimental setups.

Article Snippet: BLU0588 (MedChemExpress, CAS No.: 2810474-78-3) and H89 (MedChemExpress, CAS No.: 130964-39-5) were dissolved in DMSO and stored as a 10 mM stock solution at −80 °C until use.

Techniques: Comparison, Concentration Assay

Journal: Cells

Article Title: Neuronal Primary Cilia Mediate Noggin Release to Enable Extracellular Signaling

doi: 10.3390/cells14201607

Figure Lengend Snippet: Mature neuron activity increases noggin expression and release. ( A ) Noggin mRNA expression levels in neurons cultured 28 days in vitro treated with 20 mM KCl collected at 6 h and 24 h; n = 3, ** p < 0.01. ( B ) Representative western blot probed for noggin protein in neurons cultured 28 DIV treated with 20 mM KCl for 6 h. ( C ) Quantification of western blot probed for noggin showing levels of intracellular noggin after cultured cells were treated with 20 mM KCl for 6 h and 24 h; n = 4, **** p < 0.0001. ( D ) Quantification of extracellular noggin in the media of cultured neurons following 20 mM KCl treatment for 6 and 24 h as assessed by ELISA; n = 4, ** p < 0.01. ( E ) Representative immunocytochemistry showing 28 DIV neurons treated with 20 mM KCl and stained for noggin (green) showing ciliary distribution of noggin staining (indicated by white arrows) at 0 h of treatment, increased ciliary and cytoplasmic noggin expression at 6 h, loss of ciliary noggin distribution and diminished cytoplasmic expression of noggin at 24 h, and return to baseline ciliary distribution of noggin at 72 h. ( F ) Quantification of extracellular noggin in the media of cultured neurons following 20 mM KCl 6 h treatment. Addition of the PKA inhibitor, H89, blocks the effect of KCl depolarization on noggin release; n = 6, ** p < 0.01 Data shown as mean ± SEM. Multiple comparisons test with one-way ANOVA for multiple group experiments. ** p < 0.01; **** p < 0.0001. Scale bars: ( E ): 5 μm.

Article Snippet: The PKA inhibitor, H89 dihydrochloride (Tocris Bioscience, Bristol, UK, Cat. No. 2910) solid, was dissolved in PBS at a working concentration of 100 μM and used at a final concentration of 20 μM in prepared media.

Techniques: Activity Assay, Expressing, Cell Culture, In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, Immunocytochemistry, Staining