myh9 inhibition assay  (TargetMol)

Journal: Journal of Lipid Research

Article Title: Sphinganine as a potentially relevant metabolite in pulmonary involvement of primary Sjögren’s syndrome

doi: 10.1016/j.jlr.2025.100961

Figure Lengend Snippet: Identification of Myh9 as a direct binding target of Sa and evaluation of complex stability. A: Schematic representation of the chemoproteomic workflow using a biotin-labeled Sa probe in NIH3T3 cells. B: Coomassie blue-stained SDS-PAGE gel showing differential protein bands between treatment groups. The band marked by a dashed box was excised for LC-MS/MS analysis. C: Molecular docking model displaying the binding conformation of Sa with Myh9 and key interacting residues. D: Two-dimensional interaction diagram illustrating hydrogen bonds and hydrophobic interactions between Sa and Myh9. E–G: Molecular dynamics simulation of the Sa–Myh9 complex: (E) RMSD, (F) RMSF of chain D, and (G) Rg plots, reflecting structural stability, residue flexibility, and compactness over 100 ns. H–K: CETSA evaluating Myh9 thermal stability in A253 (H and J) and NIH3T3 (I and K) cells treated with Sa (10 μg/ml). Melting curves derived from CETSA illustrate increased thermal stability of Myh9. Data are presented as mean ± SD. P < 0.05, P < 0.01 versus control group. CETSA, cellular thermal shift assay; RMSD, root mean square deviation; RMSF, root mean square fluctuation; Rg, radius of gyration.

Article Snippet: For the Myh9 inhibition assay, cells were pretreated with 2 μM Blebbistatin (TargetMol, Shanghai, China) for 4 h in serum-free medium.

Techniques: Binding Assay, Labeling, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Residue, Derivative Assay, Control, Thermal Shift Assay

Journal: Journal of Lipid Research

Article Title: Sphinganine as a potentially relevant metabolite in pulmonary involvement of primary Sjögren’s syndrome

doi: 10.1016/j.jlr.2025.100961

Figure Lengend Snippet: Functional role of Myh9 in Sa-induced epithelial injury and fibroblast activation. A: Relative mRNA expression of Myh9 in submandibular gland tissues as measured by qRT-PCR. B: Immunohistochemical staining analysis and (C) quantitative analysis of the expression of Myh9 in lung tissues. D and E: Western blot analysis (D) and corresponding quantification (E) of Myh9 protein expression in A253 cells treated with increasing concentrations of Sa. F and G: Western blot (F) and densitometric analysis (G) of Myh9 expression in NIH3T3 cells treated with Sa or TGF-β1 (positive control). H and I: Western blot (H) and quantification (I) of AQP5 expression in A253 cells treated with Sa, with or without the Myh9 inhibitor blebbistatin. J and K: Western blot (J) and quantification (K) of fibronectin and α-SMA protein levels in NIH3T3 cells treated with Sa, with or without blebbistatin. Data are presented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001; ns, not significant. # P < 0.05, ## P < 0.01 Sa group with 4-PBA treatment. α-SMA, alpha-smooth muscle actin; 4-PBA, 4-phenylbutyric acid; AQP5, aquaporin-5

Article Snippet: For the Myh9 inhibition assay, cells were pretreated with 2 μM Blebbistatin (TargetMol, Shanghai, China) for 4 h in serum-free medium.

Techniques: Functional Assay, Activation Assay, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Western Blot, Positive Control

Journal: Journal of Lipid Research

Article Title: Sphinganine as a potentially relevant metabolite in pulmonary involvement of primary Sjögren’s syndrome

doi: 10.1016/j.jlr.2025.100961

Figure Lengend Snippet: Molecular mechanism of Sa induces salivary hyposecretion and pulmonary fibrosis in pSS through the ATF6–Myh9 signaling pathway. Sa, sphinganine; pSS, primary Sjögren’s syndrome.

Article Snippet: For the Myh9 inhibition assay, cells were pretreated with 2 μM Blebbistatin (TargetMol, Shanghai, China) for 4 h in serum-free medium.

Techniques: