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. 2018 Sep 13;11:328. doi: 10.3389/fnmol.2018.00328

Figure 4.

Figure 4

Stargazin dephosphorylation is essential to synaptic down-scaling in cortical neurons. (A) Amino acid sequences of the CT2 region of STG and phosphomimetic/phosphodead STG mutants. Serine residues that can be phosphorylated in vitro and in vivo are indicated with dots on the top row. S9A mutant mimicks the fully dephosphorylated state of stargazin, while the S9D mutant mimicks the fully phosphorylated form of stargazin. (B) Cortical neurons were transfected with stargazin (STG, WT/S9A/S9D) along with GFP at DIV 14 and treated with PTX for 4 h or 24 h. (C) Surface GluA1 was analyzed by immunocytochemistry after PTX treatment during 4 h. Scale bar 5 μm. (D) PTX induced a decrease in surface GluA1 integrated intensity in Wild-type stargazin (WT) STG and S9A-transfected neurons, but overexpression of the stargazin S9D mutant blocked PTX-induced GluA1 removal from the surface of cortical neurons. N ≥ 23, from three independent experiments. (E) Surface GluA1 was analyzed by immunocytochemistry following 24 h PTX treatment. Scale bar 5 μm. (F) Twenty-four hours PTX treatment induced a decrease in surface GluA1 integrated intensity in WT STG and S9A-transfected neurons, but overexpression of the S9D mutant blocked PTX-induced GluA1 removal from the surface of cortical neurons. N ≥ 26 from three independent experiments. Two-way ANOVA, Bonferroni multiple comparison test. In (D,F), data are presented as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001).