Checkmate mammalian two hybrid system

We collected whole cell lysates from human articular chondrocytes using an M-PER kit, and performed co-IP using a Catch and Release kit (Upstate Biotechnology, Lake Placid, NY) with anti-Flag (F7425, Sigma-Aldrich, St. Louis, MO), anti-Runx1 (23980), anti-Sox5 (94396), anti-Sox6 (30455) and anti-Sox9 (185230) antibodies (Abcam, Cambridge, UK). Immune complexes were eluted and subjected to SDS-PAGE. Mammalian two-hybrid assays were performed with the Checkmate Mammalian Two-Hybrid System (Promega, Madison, WI).

293T cells(ATCC, USA) were maintained in DMEM containing 10% fetal bovine serum (FBS) and were transiently transfected using Lipofectamine 2000 reagent (Invitrogen, USA). All mutant PPARγ plasmids were created using the Quick-Change site-directed mutagenesis kit (Stratagene, USA). Twenty-four-well plates were plated 24 hours prior to transfection (5 × 10⁴ cells per well). For the Gal4-driven reporter assays, the cells were transfected with 200 ng of Gal4-LBDs of various nuclear receptors and 200 ng of pG5Luc reporter (Promega, USA). Ligands were added five hours after transfection. Cells were harvested 24 h later for the luciferase assays. Luciferase activities were analyzed as the the instruction of CheckMate™ Mammalian Two-Hybrid System (Promega, USA).

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.