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