Immunoblotting was performed as previously described [44 (link)]. Equal amounts of tissue lysates were used for immunoblotting. Blots were incubated with specific antibodies to BMP-1 and LTBP-1 (Abcam, ab205394 and ab78294), Flag (Sigma-Aldrich, F3165), ALK5 (R&D system, MAB5871), Col3a1 and Fibronectin 1 (Novus Biologicals, NB600 and NBP1–91258). β-Actin (Sigma-Aldrich, A2228) was used as a loading control. Immunofluorescence was performed as previously described in detail [44 (link)]. We used specific antibodies to CD34 (BD Bioscience, 553731 or Abcam, ab54208), Nkx2.1 (Abcam, ab76013) and VE-cadherin (BD biosciences, 562243). The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, D9564).
Protocol detail
Immunoblotting and Immunofluorescence of BMP-1, LTBP-1, and Markers
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Actin
Alk5
Antibodies
Biologicals
Bmp 1
Dapi
Fibronectin 1
Immunofluorescence
Nkx2 1
Novus
Nuclei
Tissue
Ve cadherin
Corresponding Organization :
Other organizations : University of California, Los Angeles, Stanford University
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Variable analysis
independent variables
- Antibodies used for immunoblotting: BMP-1, LTBP-1, Flag, ALK5, Col3a1, Fibronectin 1
- Antibodies used for immunofluorescence: CD34, Nkx2.1, VE-cadherin
- Protein expression levels of BMP-1, LTBP-1, Flag, ALK5, Col3a1, Fibronectin 1
- Presence and localization of CD34, Nkx2.1, VE-cadherin
- β-Actin used as a loading control for immunoblotting
- DAPI used to stain nuclei for immunofluorescence
- No positive or negative controls were explicitly mentioned in the provided information.
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