Protein-tyrosine phosphatase expression during ES-derived EB differentiation. (A) mRNA from ES cells at different days following induction of EBs was reverse transcribed and amplified with primers 22F and 2779R after reverse transcription with primer 2R (plus oligo dT) and products observed on agarose gel (upper panel); the ES cell mRNAs were also amplified with β-actin primers and the 212 bp product was visualized with ethidium bromide to ensure that approximately equal amounts of mRNA were used for each time point, as shown in the agarose gel in the middle panel. The gel in the upper panel was blotted to a nylon membrane and hybridized to a random primed radiolabeled Ptpγ cDNA probe (lower panel); control lanes 1 through 3 contained RT-PCR product from Swiss 3T3 cell line IT22, BALBc 3T3 stably transfected with an expression plasmid for the EGFR gene, and NIH 3T3 cells stably overproducing the PLCγ gene product, respectively; lanes 4 to 12 contain the RT-PCR product from undifferentiated ES cells, ES cells 1 day after seeding in bacteriological petri dishes without LIF, 2, 3, 5, 7, 10, 14, and 21 days, respectively; lanes 13 and 14 contain the PCR product from a kidney cDNA clone and a brain cDNA clone, respectively. (B) Reverse transcripts from EB cells at 0 through 28 days of differentiation were tested for expression of two other receptor tyrosine phosphatases, Lrp which was uniformly expressed during differentiation and Lar which was not expressed at any time point; to show reproducibility of induction of expression of Ptpγ in this experiment the same RT products were amplified with the Ptpγ primers, products run on agarose, blotted and hybridized to the Ptpγ probe as in (A).