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. 2019 Jun 17;8(7):bio042283. doi: 10.1242/bio.042283

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© 2019. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 6. — Expression of ActRIIA V3 K76 variant receptors does not inhibit BMP7-evoked Smad1/5/8 phosphorylation. (A) Western blots of dsRed^ΔWEHI or AIIA^ΔWEHI lysates co-expressing ActRIIA V3 K76A incubated with or without 50 ng/ml BMP7 for 30 min and probed for pSmad1/5/8. Detection of tSmad provided a loading control. BMP7 stimulated robust increases (mean±s.e.m.) in the levels of pSmad1/5/8 in dsRed^ΔWEHI (195% over control, n=2) or AIIA^ΔWEHI (113% over control, n=2; *P<0.05, Student's two-tailed t-test) cells co-expressing ActRIIA V3 K76A. (B) Quantification of western blots (mean±s.e.m.) of dsRed^ΔWEHI or AIIA^ΔWEHI lysates co-expressing pcDNA3, ActRIIA V3, ActRIIA V3 K76A and ActRIIA V3 K76E incubated in the presence of 50 ng/ml BMP7 for 30 min and probed for pSmad1/5/8. Detection of tSmad provided a loading control. Levels of pSmad1/5/8 were normalized to pSmad1/5/8 levels in unstimulated cells. All conditions demonstrated increases in response to BMP7 stimulation compared with control cells. The data for the unstimulated controls are not shown. There was no significant difference in the level of pSmad1/5/8 in dsRed^ΔWEHI lysates compared with pSmad1/5/8 levels in AIIA^ΔWEHI cells for any of the transfection conditions.