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Figure 2 | Journal of Molecular Signaling

Figure 2

From: The Caenorhabditis elegans D2-like dopamine receptor DOP-2 physically interacts with GPA-14, a Gαi subunit

Figure 2

Interaction studies of dopamine receptor DOP-2XL with Gα protein GPA-14. (A) Split-ubiquitin based pair-wise interaction. Yeast two-hybrid screen with full-length DOP-2XL yielded GPA-14 as a positive interaction partner that was validated by pair-wise interaction between dop-2c (cloned in pBT3-STE) and gpa-14 (cloned in pPR3-N and pPR3-STE). The interaction was determined by growth assay on selective growth media and X-gal filter overlay assay. For the growth assay, selected yeast colonies were streaked onto a master plates (plate 1, SD/-Leu-Trp), and selective plates (plate 2, SD/-Ade-His-Leu-Trp) containing 1 mM 3-AT. Absence of adenine from the selection medium is useful since in the absence of a protein-protein interaction, the ADE2 reporter gene is not transcribed and therefore, a red-colored intermediate accumulates in the adenine metabolic pathway and gives red color to the colony (plate 1). To perform X-gal filter lift-off assay (plate 3, X-gal) colonies from master plate were replica transferred to 3 mM filter paper and assay was performed as described by Dualsystems. The growth results and X-gal assay results are interpreted as good interaction (+), weak interaction (+/-), or no interaction (-). (B) In-vitro interaction between DOP-2XL and GPA-14. GST-tagged GPA-14 and GPA-15 (~ 72 KDa) was purified by immobilization with paramagnetic GST particles (data not shown). DOP-XL protein (~94 KDa) expression was obtained by in-vitro transcription/translation method (lane1). DOP-2 XL was incubated either with purified GST:GPA-14 or with purified GST:GPA-15, GST, paramagnetic beads, as controls. At the end of the incubation the beads were collected from the mixtures and washed. The eluted samples were applied to SDS PAGE and transferred to PVDF membrane and probed colorimetrically. In-vitro synthesized DOP-2XL was pulled down with Magne-GST beads when incubated with GST:GPA-14 (lane 2) but not when incubated with GST alone (lane 3), or with magnetic beads only (lane 4); however, a weak band is visible with GST:GPA-15 (lane 5) which is likely due to non-specific interaction.

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