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. 2012 Aug 22;32(34):11618–11630. doi: 10.1523/JNEUROSCI.5068-11.2012

Figure 4.

Figure 4.

PICK1 restricts GluA2 trafficking to the cell surface after glycine stimulation. A, PICK1 knockdown allows the traffic of GluA2 to the plasma membrane within 5 min after glycine stimulation. Dissociated hippocampal neurons transfected with plasmids expressing EGFP alone or EGFP plus PICK1 shRNA were exposed to elevated glycine for 3 min, returned to normal medium for the specified time, and fixed 0, 5, 10, or 20 min after stimulation. Controls did not receive the stimulus. Cells were processed for immunocytochemistry as described for Figure 1. Graph shows surface/internal ratio relative to unstimulated controls. **p < 0.005, ***p < 0.001. n = 4 independent experiments, with 12–15 cells in each. Scale bar, 20 μm. B, PICK1 overexpression further restricts GluA2 trafficking to the plasma membrane in response to glycine stimulation. Dissociated hippocampal neurons transfected with plasmids expressing WT–PICK1–IRES–EGFP were treated as in A. Graph shows surface/internal ratio relative to unstimulated controls. ***p < 0.001. n = 4 independent experiments, with 12–15 cells in each. Scale bar, 20 μm. C, Coexpression of sh-resistant WT–PICK1 rescues the shRNA phenotype. Dissociated hippocampal neurons transfected with plasmids expressing shRNA against PICK1 plus sh-resistant WT–PICK1–IRES–EGFP were treated as in A. Graph shows surface/internal ratio relative to unstimulated controls. *p < 0.0125. n = 4 independent experiments, with 12–15 cells in each. Scale bar, 20 μm.