drug h89  (Tocris)

Journal: Cell Communication and Signaling : CCS

Article Title: Intestinal epithelial PIEZO1 regulates macrophage-to-myofibroblast transition via epithelial-derived TGF-α signaling in Crohn’s disease

doi: 10.1186/s12964-026-02941-w

Figure Lengend Snippet: PIEZO1-dependent epithelial signaling regulates MMT via PKA/NF-κB P65 pathway. A Western blotting was performed to detect the phosphorylation level of AKT, ERK1/2, CREB, PKA and NF-κB P65 in THP-1 macrophages with CM for 24h (n = 5). P values were calculated using Student’s t-test. B Immunofluorescence showing the expression of CD68 + p-PKA + cells or CD68 + p-NF-κB P65 + cells in colonic mucosa between healthy controls and CD patients (n = 4). P values were calculated using Student’s t-test. C Endogenous interaction between PKA and NF-κB P65 in THP-1 macrophages. Co-immunoprecipitation (Co-IP) analysis demonstrating the physical association between PKA and P65. THP-1 macrophages were stimulated with CM from Caco-2 transfected with either shCTL or shPIEZO1. Lysates were immunoprecipitated (IP) with anti-PKA antibodies or an IgG isotype control, followed by immunoblotting (IB) with anti-PKA and anti-P65 antibodies. The presence of P65 in the PKA-IP fraction confirms the formation of the PKA/P65 complex. GAPDH was used as the loading control for Input lysates. D THP-1 macrophages were treated with CM derived from Caco-2-shCTL cells at 50% or 100% confluence (denoted as 50 and 100). For the 100 condition, cells were further transfected with siNC or siP65 as indicated. The protein expression of α-SMA, COL1A2 and NF-κB P65 were analyzed by Western blotting (n = 5). P values were calculated using one-way ANOVA and Tukey’s multiple comparisons test. E THP-1 macrophages were treated with CM derived from Caco-2-shCTL cells at 50% or 100% confluence (denoted as 50 and 100). For the 100 condition, cells were further treated with the PKA inhibitor H-89 or NF-κB activator 1(activator-1), either alone or in combination, as indicated. Western blotting detected the expression of α-SMA, COL1A2 and the phosphorylation level of PKA and NF-κB P65 (n = 5). P values were calculated using one-way ANOVA and Tukey’s multiple comparisons test. Data are represented as mean ± SD, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Scale bar =50 μm. CM conditions are defined as in Fig.

Article Snippet: Differentiated THP-1 macrophages were then treated with CM collected from Caco-2-shPIEZO1 (KD) or Caco-2-shCTL (CTL) cells for 24 h. In the PKA inhibition experiment, THP-1 macrophages were treated with CM with or without H-89 dihydrochloride (MedChemExpress, USA; Cat. # HY-15979 A) (30 nmol/L) for 24 h. In the NF-κΒ P65 activation experiment, THP-1 macrophages were stimulated with CM with or without NF-κΒ activator 1 (Selleck Chemicals, USA; Cat. # E1351) (9 nmol/L) for 24 h.

Techniques: Western Blot, Phospho-proteomics, Immunofluorescence, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Control, Derivative Assay

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