nuclear export signal nes  (Addgene inc)

Journal: iScience

Article Title: Mechanosensitive interactions between Jag1 and Myo1c control Jag1 trafficking in endothelial cells

doi: 10.1016/j.isci.2025.113879

Figure Lengend Snippet: Proximity biotin labeling, mass spectroscopy, and shear platform to identify shear stress-sensitive Jag1-interacting proteins (A) The engineered ascorbate peroxidase (APEX2) converts biotin tyramide to a free radical-carrying species in the presence H 2 O 2 , allowing labeling of nearby peptides. Coupling APEX2 to Jag1 and performing labeling interaction under different mechanical loading conditions allows the identification of mechanoresponsive Jagged-interacting factors (mJIFy and mJIFz). (B) Jag1 engineered with APEX2 on the C-terminal (intracellular) domain enables labeling of cytoplasmic proximal proteins in the presence or absence of flow-induced shear stress. (C) Immunoblot of Jag1 and Lenti Jag1-APEX2 expressions. HUVECs were transduced with Lenti-Jag1-APEX2, exposed to static or shear conditions, and blotted for Jag1 and Actin. (D and E) Immunofluorescence microscopy of cells labeled with streptavidin 488 (SA488, green) and 4',6-diamidino-2-phylindole (DAPI; blue). MCF7 cells were transduced with (D) Lenti-APEX2-NES or (E) Lenti-Jag1-APEX2, biotinylated, and stained with streptavidin 488 and DAPI; scale bars, 25 μm.

Article Snippet: As control plasmid for the proximity labeling we used an APEX2 domain coupled to a nuclear export signal (NES) for cytoplasmic labeling. pcDNA3 APEX2-NES (RRID:Addgene_49386) was a gift from Alice Ting through Addgene.

Techniques: Labeling, Mass Spectrometry, Shear, Western Blot, Transduction, Immunofluorescence, Microscopy, Staining