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. 2013 Oct 22;23(5):457–466. doi: 10.1089/scd.2013.0220

FIG. 4.

FIG. 4.

Smad2 phosphorylation and Lefty1 expression in Lefty2 KD cells. (A) Phospho-Smad2 in two Lefty2 KD mESC lines under self-renewal or differentiation culture conditions was analyzed by immunoblot analysis (left panel). The band intensity of p-Smad2 was quantified by normalization against β-actin (right panel). Cells were differentiated by removing LIF and adding RA for 3 days (RA-d3). (B) Lefty2 KD was transfected with a control (Con) or a Lefty2 expression plasmid and then spontaneously differentiated for 3 days. Differentiating cells were examined by light microscopy. (C) Lefty2 KD mESCs transfected with a control (Con) or a Lefty2 expression plasmid were analyzed for phospho-Smad2 levels by immunoblot analysis. (D) Nodal expression in Lefty2 KD and control ESCs was analyzed by real-time RT-PCR. (E) Lefty1 expression in Lefty2 KD and control ESCs was analyzed by real-time RT-PCR. All values are mean±SD from at least three independent experiments. *P<0.05 and **P<0.01 based on Student's t-test analysis. Lefty2KD, stable Lefty2-knocked-down mESC line; Con, Lefty2KD transfected with control plasmid.