Figure 3. Eag80 is produced by alternative splicing of eag.

(A) Anisomycin, a translational inhibitor, inhibits the production of Eag80. 23 h after transfection, cells were incubated with anisomycin at indicated concentrations for 1 h, then for a further 5 h with or without addition of 162 nM TPA. (B) Actinomycin D, a translational inhibitor, significantly inhibits the production of Eag80. 23 h after transfection, cells were incubated with actinomycin D at indicated concentrations for 1 h, then for a further 5 h with or without addition of 162 nM TPA. (C) Reverse transcription identifies an Eag80 splice variant. mRNA was extracted from eag-transfected COS cells treated with TPA and wild type Canton-S heads. (D) Eag80 contains Eag N-terminal sequences. A full length eag cDNA with an N-terminal Myc tag was transfected into COS cells, which were then treated with or without 162 nM TPA for 5 h. Immunoblot of cell lysates were done using either anti-Myc or an anti-Eag antibody that recognizes the distal C-terminal. (E) A synthetic Δ67-698 eag cDNA produced a family of protein bands identical in size to Eag80 in the absence of TPA. Treatment of cells with TPA for 5 h did not alter the production of this protein, although it did induce production of identical bands from a full length cDNA.