Figure 6

Sig-1 receptors and its agonist, PRE084 upregulate calpastatin in PC6.3 cells. (a and b) Immunoblot and quantification. Calpastatin level was decreased in cells expressing 120Q-huntingtin and was restored by using 0.3 μM PRE084. β-actin was used as control. Values represent mean±S.D., n=3. **P<0.01 for 120Q versus C. and for 120Q-PRE084 versus 120Q. (c) Immunoblot. Expression of 120Q-huntingtin induced the appearance of the calpain-specific 145-kDa cleavage product of α-spectrin and this was reduced by 0.3 μM PRE084. Typical blot is shown and was repeated three times. (d and e) Immunoblot and quantification. Stimulation of cells for 24 h with 0.3μM PRE084 increased calpastatin levels compared with control (lanes 1–3). Overexpression of Sig1-R also increased calpastatin levels (lane 4). Values represent mean±S.D., n=3. *P<0.05 for PRE084 versus C and for Sig1-R versus C. (f) Calpastatin mRNA levels were determined in cells expressing either the 17QFL or the 75QFK huntingtin protein by quantitative PCR using specific primers for calpastatin as described in the Materials and Methods. The mutant 75QFL huntingtin decreased calpastatin mRNA and this was not significantly counteracted by PRE084. Values are means±S.D., n=3. *P<0.05 for 75QFL versus 17QFL