Figure 6

TFR regulates AP2-GluR2 binding efficiency via AP2 recruitment.

(A) Total expression level of endocytic machinery proteins in TFR KO neurons. AP2, clathrin and dynamin were imunoblotted in mice whole brain lysates and P2 fractions. Protein level changes in TFR KO neurons is measured as a ratio of KO to WT (n = 3). (B) AP2-GluR2 interaction is reduced in P2 fractions of P14 TFR KO mice brain. Antibody against GluR2 C-terminus was used in the pull-downs and then imunoblotted with AP2 antibody. (Three experiments from three pairs of WT and KO mice, Student’s t test, *p < 0.05). (C) Co-immunoprecipitation of GluR2 with AP2 (antibody against AP2 μ2 subunit) in normal and stimulated 14 div cultured neurons from WT and TFR KO mice. Endogenous GluR2 was immunoprecipitated with the anti-GluR2 antibody and the co-immunoprecipitated AP2-subunits were detected by AP2 antibody in control (normal growth medium), AMPA- (100 μM, 10 min) and NMDA- (50 μM, 10 min) treated neurons. TFR-GluR2 interaction signal cannot be detected by immunoblotting with TFR antibody (Invitrogen). (D,E) AP2 particle distribution at cell surface of 14 div neurons transfected with different TFR constructs. 14 div primary neurons were transfected with TFR-mCherry, TFR-Y20A or TFR-F23A constructs for 24 hr (non-treatment as control group) and then fixed for immunofluorescence staining with AP2, GluR1 or GluR2. Yellow arrows indicate where high magnification images taken from. Dot lines represent the cell surface, below which is the intracellular domain. Scale bar represents 20 μm in low magnification panel and 5 μm in high magnification panel. (F,G) AP2 co-localization ratio with GluR1 and GluR2. Co-localization ratio is measured as percentage of AP2-positive GluR1 or GluR2 particles and normalized non-transfection WT neurons as the control group. *P < 0.05.