Journal: Cell reports

Article Title: Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation.

doi: 10.1016/j.celrep.2016.04.066

Figure Lengend Snippet: Figure 6. PD407824 Increases BMP Sensitivity by Depleting p21 and Activating CDK9, which Then Phosphorylates the SMAD2/3 Linker Region, Leading to Decreased Levels of SMAD2/3 Protein and Enhanced Levels of Nuclear SMAD1 (A) PD407824 treatment increases the binding of SMAD1 and SMAD4 proteins indicated by co-immunoprecipitation. (B) PD407824 causes depletion of SMAD2/3 protein levels, measured by western blotting; SMAD2/3 protein levels are normalized to beta-ACTIN protein levels. (C) PD407824 induces the phosphorylation of the SMAD2/3 linker region (T179 for SMAD3). The phosphorylation level was normalized to total levels of SMAD2 protein. (D–F) Flavopiridol, a CDK inhibitor, blocks the PD407824 induced increased Id2 transcript expression (D), upregulated T179 phosphorylation of SMAD3 and decreased SMAD2/3 total protein (E), and increased binding of SMAD1 and SMAD4 proteins indicated by co-immunoprecipitation (F). (G) Knockout of CDK9 blocks the synergistic effect of PD407824. (H) PD407824 depletes p21 protein levels, shown by western blotting. The p21 protein levels are normalized to b-actin protein levels. (I) Relative Id2 transcript expression in p21+/ and p21/ cells. Uninfected cells (wild-type [wt]) and cells infected with sgRNA targeting GFP were used as negative controls. See also Figure S4.

Article Snippet: The antibodies were rabbit anti-phospho-SMAD1/5/9 (Cell Signaling Technology, 9511), rabbit anti-SMAD1 XP (Cell Signal, 6944), rabbit anti-SMAD2/3 (Cell Signal, 3102), rabbit anti-p21 (C-19 antibody; Santa Cruz Biotechnology, sc379), rabbit anti-phospho-SMAD3 T179 (Rockland, 600-401-C48S), and mouse anti-b-actin (Sigma-Aldrich, A1978).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Phospho-proteomics, Expressing, Knock-Out, Infection

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Astragaloside IV alleviates lung inflammation in Klebsiella pneumonia rats by suppressing TGF-β1/Smad pathway

doi: 10.1590/1414-431X2023e12203

Figure Lengend Snippet: A , Expression of transforming growth factor (TGF)-β1 in lung tissues in K. pneumoniae rat model treated with astragaloside IV (AS-IV) or TGF-β1 inhibitor SB-431542 detected by immunohistochemistry (scale bar 50 μm); B , Expression levels of proteins related with TGF-β1/Smad, p-Smad2/Smad2, and p-Smad3/Smad3 pathways in lung determined by western blot. Data are reported as means±SD (n=3). *P<0.05, **P<0.01 compared with Control group; # P<0.05, ## P<0.01 compared with K. pneumoniae group (ANOVA).

Article Snippet: The primary antibodies were: TGF-β1 polyclonal antibodies (1:800, SAB4502954, Sigma-Aldrich), p-Smad2 polyclonal antibodies (1:100, orb451161, Biorbyt, China), Smad2 polyclonal antibodies (1:100, orb507656, Biorbyt), p-Smad3 polyclonal antibodies (1:100, orb313112, Biorbyt), Smad3 polyclonal antibodies (1:100, orb94696, Biorbyt), NF-κB p-p65 polyclonal antibodies (1:100, orb501839, Biorbyt), NF-κB p65 polyclonal antibodies (1:100, orb344389, Biorbyt), IκBα polyclonal antibodies (1:1000, 4812, Cell Signaling Technology, China), p-IκBα polyclonal antibodies (1:800, 2859, Cell Signaling Technology), and GAPDH polyclonal antibodies (1:1000,5174, Cell Signaling Technology).

Techniques: Expressing, Immunohistochemistry, Western Blot, Control