Nodal/Activin Signaling in ES Cell Fate Decisions Figure 2. Smad2 Phosphorylation and Induction of Distinct Cell Fate Decisions Are Determined by Differential Nodal/Activin Signaling. (A) Western blot and densitometry quantitation of Smad2 phosphorylation in ES cells after 18 hours stimulation with Activin (ACT) and inhibition with SB-431542 (SB) compared to DMSO vehicle control at the indicated doses in chemically defined KSR media. The same blot was stripped and reprobed with total Smad2 and Pcna loading control antibodies. Secondary band in the total Smad2 blot corresponds to cross-reaction with total Smad3. Graph shows densitometry measurements of pSmad2 protein bands relative to total Smad2 in each treatment with the DMSO control at 100%. (B) Real-time PCR analysis of early cell fate markers in differentiated ES cells after 6 days treatment in ACT, DMSO and SB normalized to b-actin housekeeping control. Top panel shows induction of markers in ACT treatments expressed as fold change over the control KSR media. PLoS Genetics | www.plosgenetics.org 5 June 2011 | Volume 7 | Issue 6 | e1002130