<p style="text-align: justify;"><b>Southern Blot Analysis</b></p>

<p style="text-align: justify;">Southern blot analysis confirmed in the combined trait corn product MON89034 &times; TC1507 &times; MON88017 &times; DAS-59122-7 the presence of sequences identical to sequences derived from MON 89034 and MON 88017. Hybridization patterns for the combined trait product were identical to those of the parental lines with <i>cry1F, cry34Ab1, cry35Ab1</i>, and the <i>pat</i> gene probes indicating that the TC1507 and DAS-59122-7 insertions were unaffected by combining with MON89034 and MON88017 through conventional breeding.</p>

<p style="text-align: justify;"><b>Protein Expression Levels</b></p>

<p style="text-align: justify;">MON89034 x TC1507 x MON88017 x DAS-59122-7 is a combined trait corn that produces lepidopteran-active and coleopteran-active <i>Bacillus thuringiensis</i> (Bt) proteins, as well as the 5- enolpyruvylshikimate-3-phosphate synthase protein from <i>Agrobacterium</i> sp. strain CP4 (CP4 EPSPS) to confer tolerance to glyphosate herbicides and PAT to confer tolerance to glufosinate herbicides. The levels of the lepidopteran-active Cry1A.105, Cry2Ab2, Cry3Bb1 proteins and the CP4 EPSPS protein were determined in tissues from MON89034 x TC1507 x MON88017 x DAS-59122-7 plants grown at five U.S. field sites in 2006. The test also included a conventional corn as a negative control and MON89034 and MON88017 corns as positive controls. Leaf, root, and whole plant samples were collected over the growing season, as well as pollen and grain samples at the appropriate times. The samples were extracted and analyzed using enzyme linked immunosorbent assay (ELISA). The levels of the Cry1A.105, Cry2Ab2, Cry3Bb1, and CP4 EPSPS proteins in MON89034 x TC1507 x MON88017 x DAS-59122-7 corn were comparable to those in the appropriate MON88017 or MON89034 positive control.</p>

<p style="text-align: justify;">The levels of the coleopteran-active <i>Bacillus thuringiensis</i> (Bt) proteins Cry34Ab1, Cry35Ab1, and Cry1F, and the PAT protein were determined in tissues from MON89034 x TC1507 x MON88017 x DAS-59122-7 plants grown at five U.S. field sites in 2006. The test also included a conventional corn as a negative control and TC1507 and DAS-59122-7 parental event corn as positive controls. Leaf, root, and whole plant samples were collected over the growing season, as well as pollen and grain samples at the appropriate times. The samples were extracted and analyzed using ELISA. The results indicate that the levels of Cry34Ab1, Cry35Ab1, and Cry1F in MON89034 x TC1507 x MON88017 x DAS- 59122-7 were comparable to the levels produced in the appropriate TC1507 or DAS-59122-7 control corn. The level of PAT in MON89034 x TC1507 x MON88017 x DAS-59122-7 was higher in the combined trait products compared to TC1507 and DAS-59122-7, likely due to the presence of multiple copies of the <i>pat</i> gene in the stacks (one from each of the DAS parent lines).</p>
