Figure 4

AMPA Receptor Internalization is reduced in TFR KO Neurons.

(A–D) AMPA- and NMDA-induced internalization of AMPA receptor in WT or KO 14 div neurons. AMPA treatment (100 μM, 10 min) and NMDA treatment (50 μM, 10 min) induced robust internalization of GluR1 (A) or GluR2 (C) in WT neurons but less in KO neurons. Low-magnification image scale bar = 20 μm; High-magnification image scale bar = 5 μm. (B,D) Quantification of internalization index shown in (A,C). Internalization index is measured as the ratio of internalized fluorescence intensity to surface fluorescence intensity. Data represent mean ± SEM (n = 10 neurons for each condition, Student’s t test, *p < 0.05). (E) Surface AMPA receptor biotinylation in WT and KO neurons during NMDA stimulation. Surface GluR1 and GluR2 were biotinylated in 14 div neurons before NMDA stimulation (50 μM NMDA, 15 min). Normal growth medium was used as the negative control. Three independent experiments from WT and KO neurons were performed for both GluR1 and GluR2 (n = 5) (*p < 0.05; **p < 0.01; n.s, not significant). (F) Internalized AMPA receptor biotinylation in WT and KO neurons during NMDA stimulation. Surface GluR1 and GluR2 were biotinylated in 14 div neurons before NMDA stimulation (50 μM NMDA, 15 min). After a surface protein biotinylation washout, internalized GluR1 and GluR2 were extracted with NeutraAvidin for immunoblots. Three independent experiments from WT and KO neurons were performed for both GluR1 and GluR2 (n = 5) (*p < 0.05; n.s, not significant).