Fig. 1. Localisation of endogenous l-caldesmon in PDBu-induced podosomes in A7r5 vascular smooth muscle cells. A7r5 cells cultured on glass coverslips and were either unstimulated (A) or stimulated with 1 µM PDBu for 30 minutes (B). Cells were stained with antibodies raised against l-caldesmon and TRITC-phalloidin was used to label F-actin. The two images were merged (overlay) to show the relative localisation of l-cadesmon (green) and F-actin (red). (A) A7r5 cells grown in serum displayed a robust actin cytoskeleton with prominent stress fibres. (B) PDBu-stimulation induced the formation of podosomes containing punctate actin staining. (C) Reconstruction of the x-z profiles of typical podosomes in B; caldesmon co-localised with the actin-rich core of the podosome. Bars, 20 µm in A,B; 2 µm in C. (D) Western blot analysis of rat aortic smooth muscle cell lysate (lane 1) or A7r5 cell lysate (lane 2) probed with a general caldesmon antibody. Whereas aortic smooth muscle expresses the high molecular weight isoform of caldesmon (h-caldesmon), cultured A7r5 cells express the low molecular weight isoform (l-caldesmon). The molecular masses of protein standards (in kDa) are listed on the left.