FIGURE 3.

FQKLF motif targets HttN136 to the proteasome. A, HeLa cells were transfected for two rounds with RNA interference constructs against Rpt4 (+), a proteasome component, or a negative control RNA interference pool (-) and were then transfected with HttN136YFP expression plasmid in the second round. Western blots were performed against HttN136YFP, Rpt4, ubiquitin, and actin. Knockdown of Rpt4 increases the steady state of wt protein but has no effect in the context of a 3A mutation. B, 6 h of treatment of cells expressing unexpanded (Q25) or expanded (Q42) HttN136YFP peptide with 10 μm proteasome inhibitors (MG132, ALLN, and lactacystin) versus vehicle control (dimethyl sulfoxide (DMSO)) increases the steady state of wt peptide but has no effect on the 3A mutant. C, lactacystin (lact) titration increases the steady state of HttN136YFP but has no effect on the 3A mutant. D, steady state levels of unexpanded (Q25) or expanded (Q72) Htt Exon 1-YFP, which lacks the FQNLF motif, are unaffected by 6 h of proteasome inhibition. All of the gels are representative of at least three experiments. norm, normalized band intensity/actin.