Fig. 7.

Western blot analyses of Smad1 phosphorylation in Cv2^−/−;Tsg^−/− double mutant MEFs. Signals generated by infrared conjugated secondary antibodies were detected using the Li-Cor Odyssey imager. In the bottom blot, the artificial colors red and green correspond to signals detected at λ680 nm (anti-mouse secondary antibody against the αTubulin antibody used here as loading control) and 800 nm (anti-rabbit secondary antibody against the pSmad1 antibody), respectively. The infrared imager system allows quantification of the relative pSmad1 levels with respect to total protein loading and this is shown in the histograms at the bottom of the blots. (A) pSmad1 levels are reduced in Tsg^−/− and Cv2^−/−;Tsg^−/− MEFs when compared with Cv2^−/− or wild-type fibroblasts upon addition of 0.3 nM of BMP4, indicating that BMP4 signaling is impaired in the absence of Tsg (pro-BMP effect of Tsg) and that this is not affected by removal of CV2. (B) Effects of exogenous CV2 and Tsg on BMP4 signaling in Cv2^−/−;Tsg^−/− MEFs. Simultaneous addition of CV2 and Tsg proteins inhibited BMP signaling more efficiently than CV2 alone, demonstrating that Tsg promotes the anti-BMP effects of CV2. (C) Experiments in which CV2 was preincubated with Cv2^−/−;Tsg^−/− MEFs for 10 minutes and subsequently washed to remove excess CV2, leaving only CV2 bound to the cell surface. Note that the inhibition of BMP signaling was stronger in the presence of Tsg preincubated with BMP4 (anti-BMP effect of Tsg).