Fig. 3. Overexpression of TGF- receptors and Smad isoforms in endothelial cells. Cells were transiently transfected with expression constructs for wild type (wt), constitutively active (ca) and kinase-deficient (kd) forms of type I receptors ALK1 and ALK5, and Smad3, Smad1 and Smad5 isoforms. (A) For western blot analyses, whole-cell extracts were fractionated by 10% SDS-PAGE and transferred to blots. The membranes were then incubated with specific antibodies against ALK5, ALK1 and HA (epitope tag for receptor expression plasmids) and Smad3, Smad1 and Smad5. Bands show the presence of endogenous (control) and corresponding overexpressed levels of these proteins. On some occasions, for example for ALK1 and Smad1/5, multiple specific bands were detected. The housekeeping gene GAPDH was detected with an anti-GAPDH antibody for loading control purposes. (B) Immunofluorescence microscopy was used to detect endogenous (control) and overexpressed levels of Smad3, Smad1 and Smad5 in BAEC. Primary anti-Smad3 or anti-Smad1/5 were coupled to FITC-labeled secondary antibodies (green signal). Nuclei were stained with Hoechst 33342 (blue signal).