cfx manager software version 2 0  (Bio-Rad)

Journal: Nature Communications

Article Title: Endoglin as a BMP9 co-receptor in vascular endothelial cells: prodomain displacement and TGFBRII recruitment

doi: 10.1038/s41467-025-67531-9

Figure Lengend Snippet: a Superposition of the ENG-OR:BMP9 (5HZW) structure with the TGF-β1:TGFBRII (3KFD) structure by the ligands, showing ENG and TGFBRII can contact BMP9 simultaneously without any clash. Figure prepared using Pymol (The PyMOL Molecular Graphics System, Version 2.0, Schrödinger). b Effects of type II receptor knockdown on Smad1 phosphorylation induced by BMP9 in human pulmonary arterial endothelial cells (HPAECs). Cells were serum-starved in EGM-2, 0.1% FBS overnight, and treated with BMP9 for 15 min before being harvested for immunoblotting against an anti-pSmad1 antibody. GAPDH was used as the loading control. N = 4 independent siRNA experiments using the HPAECs from two donors. The quantification of pSmad1 relative to siCP control is shown on the right. c RT-qPCR examining the efficiency of siRNA in ( b ) and expression of other type II receptors after siRNA treatment. For ( b and c ), means ± SEM are shown, one-way ANOVA with Dunnett’s post-tests to compare with siCP-treated samples, for each receptor ( c ). Only significant p -values (<0.05) are shown. d Schematic description of the ENG pull-down assay. NHS = N-Hydroxysuccinimide, here refers to the HiTrap NHS-activated high-performance column. e Unique p e ptide counts from the mass spectrometry of the ENG pull-down assay. Only selected target proteins and controls are shown. The sequences of the peptides are mapped onto the protein sequences, as shown in ( f ) for TGFBRII and Supplementary Fig. . g Immunoblots showing Smad1/5 phosphorylation status of the samples used in the mass spectrometry experiments. h Diagrams (generated using BioRender) summarising the components investigated and the protein-protein interactions identified in the pull-down/mass spectrometry experiments. HAOECs human aortic endothelial cells, HUVECs human umbilical vein endothelial cells.

Article Snippet: The cDNAs were analysed by qPCR, and calculations were performed using the CFX Manager software version 2.0 (Bio-Rad).

Techniques: Knockdown, Phospho-proteomics, Western Blot, Control, Quantitative RT-PCR, Expressing, Pull Down Assay, Mass Spectrometry, Generated, Protein-Protein interactions