Shp-2 is necessary for brachyury and flk-1 expression in EBs. (A) Total cellular RNA was prepared from day 3, 4, and 5 EBs, reverse transcription was performed using 2 μg (1 ×), 200 ng (.1 ×), or 20 ng (.01 ×) as template, and amplification of brachyury, flk-1, and the constitutively expressed β-actin was conducted for 10, 20, or 30 cycles. PCR products were electrophoresed on agarose gels and stained with ethidium bromide for visualization. (B) Brachyury and flk-1 band intensities (1 ×, 30 cycles of amplification) were quantitated by densitometry using NIH Image and normalized to β-actin band intensities (0.01 ×, 30 cycles of amplification).