Figure 3
Forskolin and GPCR agonists induce nuclear translocation of CRTC2 and CRTC3. VSMC were transfected with GFP-CTRC1, 2 or 3 by electroporation and after 24 h were serum starved for 24 h. Cells were stimulated for 1 h with 0.5 µM forskolin (A and B, n = 3), 100 µM adenosine (n = 4; C,D), 5 µg/ml BAY60-6583 (BAY, n = 4; C,D), or 1 µM Cicaprost (n = 4; C,D). Cells were analysed for cellular localisation of CRTC proteins by fluorescence microscopy and image analysis. Data were expressed as % of cells with predominantly nuclear CRTC protein. VSMC (n = 6) serum starved for 4 h were treated for 20 or 60 min with 5 µg/ml BAY, 1 µM Cicaprost (CICA) or 10 µM forskolin (FSK). Cells were subjected to cytosolic and nuclear fractionation and proteins were subjected to SDS-PAGE and western blotting for CRTC2, CRTC3, Lamin A/C and GAPDH (E and F). CRTC2 and CRTC3 levels were quantified by densitometry and data were expressed relative to FSK (nucleus, G) or CONT (cytosol, H). *Indicates p < 0.05, ***indicates p < 0.001 relative to control; one-way repeated measures ANOVA with Student Newman Keuls post-hoc tests.
