Presynaptic UPS reporters (SNAP25-GFPu/-RFP) reflect UPS activity in cultured cells. (A) Western blot showing a significant increase in SNAP25-GFPu, but not SNAP25-RFP, in HEK293 cells after inhibition of proteasomal activity by MG132 (10 μM), ALLN (10 μg/ml), or lactacystin (lact,10 μM) for 8 h. The ratio (mean + SEM, n = 3) of GFPu to RFP was quantified (right). (B and C) In cortical or striatal neurons coinfected with adenoviral SNAP25-RFP and SNAP25-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 SNAP25-GFPu in the synapses (arrows) of cultured cortical neurons. Bars, 2.5 μm. (E) The ratio (mean + SEM n = 45) of GFPu/RFP in the cell body or the synapses was analyzed. The ratio of GFPu to RFP signal was significantly increased after inhibiting the proteasome with 10 μM MG132. *, P < 0.05; **, P < 0.01. ***, P < 0.001 as compared with control.