Journal: bioRxiv
Article Title: The YAP/TAZ antagonist, PTPN14, stabilizes SMAD4 through direct interactions in endothelial cells: Implications for Hereditary Hemorrhagic Telangiectasia
doi: 10.1101/2021.09.29.462397
Figure Lengend Snippet: A: Sub-confluent HUAECs were transfected with specified siRNAs , protein lysates were harvested 48 hours later, following the indicated treatments for 0, 1 or 24 hours with BMP9 (2ng/ml), and assayed by Western blot (left) with B: Histograms (right) showing quantification of proteins relative to siNT -transfected HUAECs. C: Schematic of BMP9-ALK-1-SMAD4 signaling. D&E : Western blot from HUAEC treated with 2ng/ml of BMP9 for 24h using siNT compared to siPTPN14 showing the effect of PTPN14 on Endoglin and SMAD4 level with the quantification graph ( E ). F : siRNA-treated cells Western blot of HUAECs cell lysates after 1h treatment with TGFβ (2ng/ml) or BMP9 (2ng/ml), with or without 5uM K02288 (an ALK-1 inhibitor) or 5uM SB431542 (a TGFβR1 inhibitor) with the observation of SMAD4, pSMAD1 and total Smad1 level trough the different conditions. G: Co-immunoprecipitation of endogenous PTPN14, SMAD4, Endoglin, ALK-1 SMAD7, or IgG control, shows interaction between endogenous PTPN14 and SMAD4,but not with Endoglin, ALK-1 and, SMAD7. H: SMAD4 pulldown with SMAD4, TAZ, pTAZ, pSAMD1/5, and SMAD7 is showing the interaction of SMAD4 with the different proteins.
Article Snippet: BMP9 signaling inhibition was performed using an ALK-1 kinase small molecule inhibitor K02288 5 μM (#S7359, Selleckchem®).
Techniques: Transfection, Western Blot, Immunoprecipitation, Control