Figure 3

Design of Tat-peptides for blocking mGlu1 receptor-caveolin interaction and its efficacy. (A) Fusion synthetic peptides encoding Tat-blocking peptide and Tat-mutant peptide are shown. Tat-mutant peptide was generated by a dual amino acid mutation (underlined) in the caveolin binding motif (609-617aa). aa, amino acid. X and ψ indicate any amino acid and aromatic amino acid, respectively. (B) Validation of incorporation of Tat-blocking peptide/mutant peptides into hippocampal primary neurons. Cells incubated with both peptides for 45 mins were stained with anti-Tat antibody and imaged with confocal microscopy. The small dots stained with anti-Tat antibody are cell debris that may not affect our analytic measurements with co-localization study and Ca²⁺ imaging. The scale bar indicates 5 μm. (C) Co-immunoprecipitation assay of caveolin and mGlu1 receptor in hippocampal primary neurons after incubation with both peptides for 45 mins. Cell lysates were immunoprecipitated with anti-caveolin antibody and immunoblotted with anti-mGlu1 receptor antibody. Quantification was shown below. The values represent relative average intensity normalized by control value ± SEM. ANOVA with posthoc test, n.s., non-significant, ***, P < 0.001 (n=3).