nicd  (Cell Signaling Technology Inc)

Journal: Sensors (Basel, Switzerland)

Article Title: Endogenous γ-Secretase Is Linked to Phagocytic Activity in Microglial Cells

doi: 10.3390/s25113298

Figure Lengend Snippet: The N100 Y-T biosensor reports γ-secretase activity in BV-2 microglial cells: ( A ) schematic presentation of the N100 Y-T biosensor and its cleavage by γ-secretase. ( B ) Western blotting using HA and cleaved Notch1 antibodies. HA antibody captures both full-length N100 Y-T and γ-secretase cleavage product: Notch intracellular domain (NICD) Y-T. In contrast, the cleaved Notch1 antibody only recognizes NICD Y-T, showing its reduction by γ-secretase inhibitors: DAPT (1 µM), L-685,458 (1 or 5 µM) for 16 h of treatment. ( C ) Spectral FRET analysis shows that DAPT (1 µM) treatment significantly increases YPet/mTurquoise-GL emission ratio (Y/T ratio). Scale bar 20 µm, N = 60 cells, Mann–Whitney U test, **** p < 0.0001.

Article Snippet: An anti-APP C-terminus antibody was purchased from BioLegend (San Diego, CA, USA), anti-β-actin was from MilliporeSigma (Burlington, MA, USA), anti-cleaved Notch1 (Val1744) and anti-GAPDH antibodies were from Cell Signaling Technology, Inc (Dover, MA, USA), and anti-HA antibody was from Abcam (Cambridge, UK). γ-Secretase inhibitors DAPT and L-685,458 were purchased from Abcam and DMSO was from MilliporeSigma. pHrodoTM Red Escherichia coli ( E. coli ) BioParticlesTM, pHrodoTM Green Zymosan BioParticlesTM, and LysoTrackerTM Deep Red were from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Activity Assay, Western Blot, MANN-WHITNEY