Suppl. Figure 1. NF-κB. gastrocnemius. WT mdx dko. mdx. pelk1. pets2. Ras. Ets2. praf. pp38. pmek-1. p38 SOS. p90 Rsk
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1 Suppl. Figure 1 NF-κ p65 gastrocnemius phase merge D pets2 dko E pelk1 dko Ets2 Ras pp38 p38 praf pmek-1 SOS p90 Rsk
2 Suppl. Figure 2 pair 1 pair 2 NF-# pair1 pair 2 p65 p50 Oct-1 ;p50+ / - D dys.!-sg "-DG dysf.
3 ;IKKβ F/F ;IKKβ F/F;ML-re E-MyH IgG positive fibers Suppl. Figure β K ;IK /F dx βf m K ;IK dx m F F/ Pax7 GPDH MyH E ;IKKβ F/F IK K βf /F IK ML Kβ F/ F - re dx D m IKKβ F/F;ML-re W T IKKβ F/F re - L ;M
4 Suppl. Figure 4 procion orange ND(mut) ND(wt) Injured fibers (%) ND(mut) ND(wt) tibialis * ND(wt) ND(mut)
5 Peptide synthesis!d peptide *as generated using an I 430 solid-phase peptide synthesizer (pplied iosystems> Foster ity> a.) *ith standard t (tertbutyloxycarbonyl)-chemistry. The peptides *ere cleahed from the resin and deprotected using hydrofluoric acid. The resulting crude materials *ere purified by HPL on a Mydac (Mydac Separations> Hesperia> a) 1P preparatihe column using gradients of acetonitrile in 0.10% trifluoroacetic acid. Follo*ing lyophilization of the purified fractions> the expected molecular *eight of the peptides *ere confirmed using matrix-assisted laser desorption ionization mass spectrometry. Primers for Real-Time PR DRP> 5T-GGGGTGGTT-3T for*ard> 5T-TGGGGG GG-3T rehersev lysozyme> 5T-GTGGGTTTGGTGT-3T for*ard> 5T- TTGGTTTTGGTGTGT-3T rehersev T!F!> 5T-GGGTGGG TT-3T for*ard> 5T-TGGTTGGTG TGG-3T rehersev IL1#> 5T- GTGGGGTGTT-3T for*ard> 5T-GGTGGGTTTTT GTG-3T rehersev MP-1V 5T-GGTGGT GGTTG-3T for*ardv 5T- GTGGTTGGGGTG-3T rehersev R!TES> 5T-TTGG TGG-3T for*ardv 5T-TTGGTG G-3T rehersev and MIP- 1!> 5T-GTTTTGGT-3T for*ardv 5T-GGGTGTGGTTT -3T reherse. GPDH primers 5T-GTT GGGTG-3T
6 for*ard> 5T-GGTTTTG-3T reherse *ere used as internal controls. Supp. Fig. 1. haracterization of signaling pathways in muscles. EMSs *ere performed from nuclear extracts prepared from gastrocnemius muscles harhested from 5-*[-old *t and mice. Immunostaining *as performed for total pr5 comparing gastrocnemius sections from 7-*[-old or mice. Scale bar denotes 50 &m.. Differential interphase contrast of a muscle section from 4-*[-old mice (left panel) or a merged image *ith ppr5 (green) and DRP (red). D and E. Western blot analysis *as performed on tibialis muscle lysates prepared from the same mice as in () probing for Ets-2 and phospho-ets2 (antibodies [indly prohided by M. stro*s[i)> p3p (1^500> Santa ruz)> phospho-p3p (1^500> ell Signaling)> phospho-el[-1> phospho-raf> ME_- 1> and p90 Rs[ (1^1000> ell Signaling)> Ras (1^500> albiochem)> and SS (1^500> apstate). Supp. Fig. 2. Effects on pathology in p65 and p50 heterozygous mice.. EMSs *ere performed from nuclear extracts prepared from tibialis muscles harhested from 2 pairs of 5-*[-old ;p50 +/+ and ;p50 +/- mice and ;p65 +/+ and ;p65 +/- mice.. Gastrocnemius muscle sections from ;p50 +/+ and ;p50 +/- mice *ere immunostained *ith F4/P0. Scale bar denotes 50 &m.. Real time PR analysis for lysozyme and DRP expression *as performed on gastrocnemius muscles.. Western blot analysis of DG
7 associated proteins *ere performed comparing *t> ;p65 +/+ > and ;p65 +/- mice. Supp. Fig. 3. Effects of muscle specific deletion of IKK# in and wild type mice.. E-MyH staining in ;IKK# F/F and ;IKK# F/F ;ML-re gastrocnemius muscles.. duantitation analysis of IgG positihe fibers from muscles used in.. HeE stained gastrocnemius cryosections from 4-*[-old IKK# F/F and IKK# F/F ;ML-re mice (nf3). Scale bar denotes 50 &m. D. Western blot analysis of tibialis anterior muscle lysates from mice used in () to probe for Pax7 and $-tubulin. E. D34 + > Sca-1 - flo* sorted cells from 4 *[-old ;IKK# F/F mice *ere differentiated and stained for MyH. Scale bar denotes 100 &m. Supp. Fig. 4. ND treatment in mice ameliorates dystrophic phenotype. and. Soleus muscles from 4-*[-old mice *ere treated *ith either *t or mut!d. Dissected muscles *ere incubated *ith 0.2% procion orange (Sigma) in _rebts Ringer solution for 1 hr. Muscles *ere *ashed> frozen> sectioned and then Hie*ed by fluorescence microscopy to guantitatihely measure myofiber inhury (> asteris[ denotes pi0.05).. HeE analysis of tibialis anterior muscles harhested from 50-day-old mice inhected *ith either mut or *t!d peptide. Scale bar in and denote 50 $m.
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