Fig. 2. Co-expression of BRII-myc and HA-BRIa-ca leads to an increase in stimulation of the Col2-luc reporter gene activity compared with the single transfection of BRIa-ca or BRII. Co-transfection of BRIa-ca with BRII-KR does not lead to an increase in luciferase activity indicating that the kinase activity of BRII is required for signalling. Limb mesenchymal cells (isolated from day 11.5 mouse embryos) were transfected with a Col2-luc reporter, and expression constructs for BRII, BRIa-ca and pRLSV40 for normalisation. Cells were plated at high density in the wells of a 24-well tissue culture dish, and 48 hours following transfection, cells were lysed and luciferase activity measured. The Col2-luc reporter is derived from the promoter and enhancer regions of the type 2 collagen gene and provides a readout on the status of chondroblast differentiation. BMPs as well as BRIa-ca are known to promote chondrogenesis in limb mesenchymal cells.