Interaction of proteins was determined using a modification of the Checkmate mammalian two-hybrid system (Promega). Two vectors were used, pAct and pBind3-D. pAct (Promega) contains the herpes simplex virus VP16 activation domain followed by a multiple cloning site. pBind3-D [64 (link)] is a modification of pBind (Promega) in which the DNA-binding domain of the yeast GAL4 gene followed by an altered multiple cloning site and the vector lacks the Renilla luciferase module.
N2a cells were seeded on 96-well plates and transfected in parallel with pAct, pBind3-D, peGFP (Clontech), and a pG5luc (Promega) expressing firefly luciferase under control of GAL4 [64 (link)]. The next day fluorescence generated by eGFP was determined for normalization and light emission generated by luciferase activity was detected after adding Bright-Glo (Luciferase Assay System, Promega) with a Multilabel Counter (PerkinElmer). Luciferase activity was normalized to eGFP-fluorescence. All transfections and analysis were performed in septuplicate and experiments repeated three times. Average relative luciferase light units and S.D. were determined using the Prism software.
N2a cells were seeded on 96-well plates and transfected in parallel with pAct, pBind3-D, peGFP (Clontech), and a pG5luc (Promega) expressing firefly luciferase under control of GAL4 [64 (link)]. The next day fluorescence generated by eGFP was determined for normalization and light emission generated by luciferase activity was detected after adding Bright-Glo (Luciferase Assay System, Promega) with a Multilabel Counter (PerkinElmer). Luciferase activity was normalized to eGFP-fluorescence. All transfections and analysis were performed in septuplicate and experiments repeated three times. Average relative luciferase light units and S.D. were determined using the Prism software.