Cleavage of Ths can be prevented through deletion of internal amino acids. (A) Thisbe amino acid sequence deleted in ThsΔ261-333Myc, including 5 potential cleavage sites, underlined in red; below the dotted black line are the amino acids that differ between the two deletion constructs, ThsΔ261-333Myc and ThsΔ261-356Myc, including 1 additional potential cleavage site, underlined in red. (B) Western blot of anti-HA immunoprecipitations from supernatant of cells transfected with pUASt-empty, HA-Ths-Myc, HA-ThsΔ261-333Myc, and HA-ThsΔ261-356Myc. HA-Ths-Myc was loaded 5x less than the other samples to equalize the exposure while resolving the double-band at 35 kD. ThsΔ261-333Myc (lane 3) is still partially cleaved but has increased full-length protein and (lane 4) ThsΔ261-356Myc has less cleavage and more full-length product, as compared to HA-Ths-Myc (lane 2). (C) 69B-GAL4 driving ThsΔ261-356Myc results in more Eve-positive cells in every hemisegment, as compared to wild-type. (D) ZenKr GAL4 driving ThsΔ261-356Myc results in extra Eve-positive cells outside the source of expression. (E) Eve-positive cells counted in 11 hemisegments for 25 embryos and averaged as in Fig. 5. The gray box represents the source of expression supported by ZenKr-GAL4. As compared to HA-Ths-Myc, ThsΔ261-356Myc has a decreased gradient of functional output, resulting in a flatter profile.
Tulin and Stathopoulos BMC Developmental Biology 2010 10:83 doi:10.1186/1471-213X-10-83