Figure 8: Smart is a novel ubiquitin ligase required for denervation-dependent atrophy.

(a) Co-immunoprecipitation experiment showing that SMART is a F-box protein that forms a SCF complex. C2C12 muscle cell lines were transfected with SMART, Skp1, Cul1 and Roc1 expression plasmids. After 24 h, cells were lysed and immunoprecipitation against FLAG-tag or control IgG was performed. Western blots for the different SCF components are shown. (b) RNAi-mediated knockdown of SMART revealed by quantitative RT–PCR (qRT–PCR). Adult TA muscles were transfected with bicistronic expressing vectors that encode either oligo 4 or scramble and GFP. Two weeks later TA muscles were collected, RNA extracted and endogenous SMART, MUSA1, Atrogin1, MuRF1 and Fbxo31 expression were analysed by qRT–PCR, n=4. (c) Inhibition of SMART prevents muscle atrophy in denervated muscles. Adult muscle fibres were co-transfected with bicistronic expressing vectors that encode shRNAs against SMART (oligo 4) or scramble and GFP and denervated. Two weeks later cross-sectional area of transfected fibres, identified by GFP fluorescence, was measured. n=6 muscles for each group. (d) Densitometric quantification of polyubiquitinated proteins in muscle extracts transfected with shRNAi against SMART or scramble. Values are normalized to GAPDH and expressed as fold increase of fed control mice. n=3 muscles for each group. Data are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student’s t-test). con, control; den, denervated; IB, immunoblotting.