Journal: Brain research bulletin
Article Title: The decrease of GluN2B and its phosphorylation at Tyr-1336 in extrasynaptic subunits is associated with neuroprotection induced by hypoxia preconditioning.
doi: 10.1016/j.brainresbull.2025.111400
Figure Lengend Snippet: Fig. 1. Effects of hypoxia preconditioning on GluN2B, pY1252 GluN2B, and pY1336 GluN2B levels in the mouse hippocampus. A: The mRNA expression level of GluN2B (n = 6 per group);B: Representative Western blotting results of GluN2B, pY1252 GluN2B, and pY1336 GluN2B (n = 6 per group);C-E: Semi-quantitative analysis showing the protein changes in GluN2B, pY1252 GluN2B, and pY1336 GluN2B;F-H: Immunofluorescence imaging and relative fluorescence intensity of GluN2B, pY1252 GluN2B, and pY1336 GluN2B in the mouse hippocampus on day 0 (n = 5 per group). Immunofluorescence imaging (300 dpi) was acquired using a laser scanning confocal microscope (A1; Nikon) with 40x magnification. Scale bar= 100μM.* p﹤0.05 versus N group; # p﹤0.05 versus H group. N: Normoxia; H: Hypoxia; HPC: Hypoxia Preconditioning.
Article Snippet: The membranes were incubated overnight with the following primary antibodies: (GluN2B (Cell Signaling Technology, #4207), p-Y1336 GluN2B (Phosphosolutions, #p1516–1336), p-Y1252 GluN2B (Phosphosolutions, #p1516–1252), spectrin (Santa Cruz Biotechnology, sc-48382), caspase-3(Cell Signaling Technology, #9662), β-actin (Santa Cruz Biotechnology, sc-47778), PSD95 antibody (Cell Signaling Technology, #3450),GluN1 antibody (Cell Signaling Technology, #5704),GluN2A antibody (Cell Signaling Technology, #4205),EEA1 antibody (Cell Signaling Technology, #2411),Rab11 antibody (Cell Signaling Technology, #5589), p97 ATPase antibody (Cell Signaling Technology, #2649)), and then incubated with secondary antibodies for 1 h. The blots were detected using ECL ultrasensitive luminescence solution (Zhang et al., 2019).
Techniques: Expressing, Western Blot, Immunofluorescence, Imaging, Fluorescence, Microscopy