Figure 5.
α-PALNRF-1is required for proteasome gene induction in vivo. (A and B) Proteasome gene expression was measured by RT-PCR analysis of mRNA preparations from innervated and 3 days (A) or 10 days (B) denervated muscles expressing a dominant negative form of FOXO3 (FOXO3ΔC) or shLacz. FOXO3ΔC expression blocked the induction of two proteasome subunit genes (Rpn6 and Rpn9) at 10 days but had no effect on proteasome gene induction at 3 days. Means ± SEM are presented as a ratio to WT innervated. N = 4 mice per condition. *, P < 0.05 versus innervated shLacz; #P < 0.05 versus denervated shLacz by one-tailed unpaired Student’s t test. (C and D) Whether or not FOXO3 is inhibited, the content of active assembled proteasomes does not change in denervated muscles. Measurement of proteasome content by native gels and immunoblotting, and proteasome peptidase activity by LLVY-AMC hydrolysis. Innervated muscles and ones denervated for 3 days (C) or 10 days (D) expressing shLacz or FOXO3-DN were analyzed. (E and F) TA muscles were electroporated with a plasmid encoding α-PALNRF-1-dominant negative (α-PALNRF-1-DN) or shLacz and mRNA preparations from transfected muscles were analyzed by RT-PCR and specific primers at 10 days (E) or 3 days (F) after denervation. Means ± SEM are presented as a ratio to innervated. N = 5 mice per condition. *P < 0.05 versus innervated; #, P < 0.05 and ##, P < 0.001 versus denervated shLacz by one-tailed unpaired Student’s t test. Blots show FLAG-α-PALNRF-1-DN expression in transfected muscles using anti-flag. (G) Inhibition of α-PALNRF-1 by the overexpression of α-PALNRF-1-DN reduces atrophy of denervated muscles. Mean ± SEM weights of denervated TA muscles are depicted as percent of innervated. N = 5 mice per time point. *, P < 0.05 and **, P < 0.005 versus innervated shLacz; #, P < 0.05 versus denervated shLacz by one-tailed unpaired Student’s t test. Source data are available for this figure: SourceData F5.

α-PAL NRF-1 is required for proteasome gene induction in vivo. (A and B) Proteasome gene expression was measured by RT-PCR analysis of mRNA preparations from innervated and 3 days (A) or 10 days (B) denervated muscles expressing a dominant negative form of FOXO3 (FOXO3ΔC) or shLacz. FOXO3ΔC expression blocked the induction of two proteasome subunit genes (Rpn6 and Rpn9) at 10 days but had no effect on proteasome gene induction at 3 days. Means ± SEM are presented as a ratio to WT innervated. N = 4 mice per condition. *, P < 0.05 versus innervated shLacz; #P < 0.05 versus denervated shLacz by one-tailed unpaired Student’s t test. (C and D) Whether or not FOXO3 is inhibited, the content of active assembled proteasomes does not change in denervated muscles. Measurement of proteasome content by native gels and immunoblotting, and proteasome peptidase activity by LLVY-AMC hydrolysis. Innervated muscles and ones denervated for 3 days (C) or 10 days (D) expressing shLacz or FOXO3-DN were analyzed. (E and F) TA muscles were electroporated with a plasmid encoding α-PALNRF-1-dominant negative (α-PALNRF-1-DN) or shLacz and mRNA preparations from transfected muscles were analyzed by RT-PCR and specific primers at 10 days (E) or 3 days (F) after denervation. Means ± SEM are presented as a ratio to innervated. N = 5 mice per condition. *P < 0.05 versus innervated; #, P < 0.05 and ##, P < 0.001 versus denervated shLacz by one-tailed unpaired Student’s t test. Blots show FLAG-α-PALNRF-1-DN expression in transfected muscles using anti-flag. (G) Inhibition of α-PALNRF-1 by the overexpression of α-PALNRF-1-DN reduces atrophy of denervated muscles. Mean ± SEM weights of denervated TA muscles are depicted as percent of innervated. N = 5 mice per time point. *, P < 0.05 and **, P < 0.005 versus innervated shLacz; #, P < 0.05 versus denervated shLacz by one-tailed unpaired Student’s t test. Source data are available for this figure: SourceData F5.

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