pcdna3 apex2 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

Journal: bioRxiv

Article Title: A proximity labeling approach to identify proteins that associate with synaptonemal complex components in Drosophila melanogaster females

doi: 10.1101/2025.10.14.682398

Figure Lengend Snippet: (A) Shown is the current model of the D. melanogaster SC. (B) Top view model of the D. melangaster SC with a schematic of the proximity ligation by APEX2::C(2)M and Cona::APEX2. APEX2::C(2)M and Cona::APEX2 incorporate into the LE and central region of the SC. In the presence of biotin-phenol and H2O2 APEX2 transfers biotin to electron-rich amino acids on nearby proteins. Nuclei are isolated from biotin-phenol treated ovaries and the biotinylated proteins were isolated on streptavidin coated beads for mass spectrometry analysis. Adapted from ( Cahoon et al . 2017 ).

Article Snippet: APEX2 was PCR amplified from pcDNA3 APEX2-NES (Addgene Plasmid #49386) without both the stop codon and nuclear export signal with NotI and NdeI restriction sites.

Techniques: Ligation, Isolation, Mass Spectrometry

Journal: bioRxiv

Article Title: A proximity labeling approach to identify proteins that associate with synaptonemal complex components in Drosophila melanogaster females

doi: 10.1101/2025.10.14.682398

Figure Lengend Snippet:

Article Snippet: APEX2 was PCR amplified from pcDNA3 APEX2-NES (Addgene Plasmid #49386) without both the stop codon and nuclear export signal with NotI and NdeI restriction sites.

Techniques: Expressing, Construct

Journal: bioRxiv

Article Title: A proximity labeling approach to identify proteins that associate with synaptonemal complex components in Drosophila melanogaster females

doi: 10.1101/2025.10.14.682398

Figure Lengend Snippet: Ovaries underwent biotin-phenol labeling followed by immunofluorescence with an antibody against C(3)G (SC, magenta), strevadin-488 (recognizes biotin, yellow), and DAPI (DNA, cyan). Germaria from (A) w 1118 and females expressing APEX2 did not show similar localization patterns between streptavidin-488 and C(3)G. In APEX2::c(2)M (C) and cona::APEX2 (D) germaria streptavidin localized in a pattern similar to full-length SC in a subset of nuclei. Boxed regions in the first column are shown enlarged without DAPI. Images are projections from z-stacks. Scale= 10 µm in germaria images and 2 µm in enlarged nuclei images. Full genotypes are w 1118 , y w; pUASp-APEX2/+; nanosGAL4::VP16/+; spa pol /+ , y w;; pUASp-APEX2::c(2)M; nanosGAL4::VP16/+; spa pol /+ , and y w; pUASp-cona::APEX2/+; nanosGAL4::VP16/cona f04903 ; spa pol /+ .

Article Snippet: APEX2 was PCR amplified from pcDNA3 APEX2-NES (Addgene Plasmid #49386) without both the stop codon and nuclear export signal with NotI and NdeI restriction sites.

Techniques: Labeling, Immunofluorescence, Expressing