Figure 3. Postsynaptic UPS reporters (PSD95-GFPu and PSD95-RFP) reflect UPS activity in cultured cells. (A) Western blot showing a significant increase in PSD95-GFPu, but not PSD95-RFP, in HEK293 cells after inhibition of proteasomal activity by MG132 (10 μM), ALLN (10 μg/ml), or lactacystin (lact, 10 μM). The ratio (mean + SEM, n = 3) of GFPu to RFP is quantified (right). (B and C) In cortical or striatal neurons coinfected by adenoviruses of PSD95-RFP and PSD95-GFPu, the expression ratio of GFPu to RFP in synapses was increased after addition of MG132. Arrows indicate synapses. Bars, 5 μm. (D) Higher magnification showing the expression of PSD95-GFPu in the synapses (arrows) of cortical neurons. Bars, 2.5 μm. (E) The ratio (mean + SEM, n = 40) of GFPu to RFP signal was significantly increased after inhibiting the proteasome by 10 μM MG132. *, P < 0.05; **, P < 0.01. ***, P < 0.001 as compared with control.
Figure 3.

Postsynaptic UPS reporters (PSD95-GFPu and PSD95-RFP) reflect UPS activity in cultured cells. (A) Western blot showing a significant increase in PSD95-GFPu, but not PSD95-RFP, in HEK293 cells after inhibition of proteasomal activity by MG132 (10 μM), ALLN (10 μg/ml), or lactacystin (lact, 10 μM). The ratio (mean + SEM, n = 3) of GFPu to RFP is quantified (right). (B and C) In cortical or striatal neurons coinfected by adenoviruses of PSD95-RFP and PSD95-GFPu, the expression ratio of GFPu to RFP in synapses was increased after addition of MG132. Arrows indicate synapses. Bars, 5 μm. (D) Higher magnification showing the expression of PSD95-GFPu in the synapses (arrows) of cortical neurons. Bars, 2.5 μm. (E) The ratio (mean + SEM, n = 40) of GFPu to RFP signal was significantly increased after inhibiting the proteasome by 10 μM MG132. *, P < 0.05; **, P < 0.01. ***, P < 0.001 as compared with control.

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