Adenoviruses were kindly supplied by BM Spiegelman. Electroporation of muscle Plasmids encoding shRNA constructs had been electroporated into muscle fibers as described before. Briefly, soleus or TA muscle of anesthetized mice was exposed and injected with 10 to thirty ul of the mixture containing the respective shRNA plasmid as well as a plasmid coding for NLS GFP. The fascia along with the skin have been sutured and also the electroporation was carried out utilizing an ECM 830 electroporation process. Eight pulses lasting 20 ms with all the frequency of 1 Hz and also the voltage set to 180 V/ cm were utilized. Mice were analyzed 4 to six weeks soon after electroporation. Denervation, nerve crush and overloading Mice have been anesthetized with ketamine and xylazine by intra peritoneal injection and sur gery was carried out underneath aseptic conditions.
For denerv ation, a segment on the sciatic nerve on the mid thigh degree dig this was excised. To induce muscle re growth, the nerve was crushed with no five Dupont forceps for ten seconds at mid thigh. To induce muscle hypertrophy, a functional overload of plantaris muscle was launched by surgical elimination of soleus and gastrocnemius muscles. Surgical treatment was performed on 1 leg only. The plantaris muscle in the contralateral leg served as manage. Antibodies The antibodies employed were in the following sources, rabbit polyclonal antibodies directed to 4E BP1 from Zymed, these recognizing Phospho 4E BP1, PKB/Akt, mTOR, S6 Riboso mal Protein or Phospho S6 Ribosomal Protein have been all from Cell Signaling Technology Inc, those towards FoxO1a were from Abcam plc, these against TSC1 have been from Bethyl Laboratories.
Rabbit monoclonal antibodies experienced directed against Phospho Akt, IRS one, FoxO3a and phospho FoxO1 /FoxO3a have been from Cell Signaling Technologies Inc. Mouse monoclonal antibodies to actinin were bought from Sigma and antibodies against HA from Covance Inc. Rat monoclonal antibodies directed towards the Laminin B2 Chain had been from Chemicon and offered by Millipore AG. The TSC2 antibodies employed had been described elsewhere. Mouse monoclonal antibodies against myosin heavy chain, slow, IIa/IIx and IIb have been obtained from the Developmental Research Hybridoma Bank. Antibodies to puro mycin had been a kind gift of Dr. Philippe Pierre. Histology and immunohistochemistry Muscle groups frozen in liquid nitrogen cooled isopentane had been reduce into 12 um cross sections. Cross sections had been fixed with 2% paraformaldehyde and perme abilized with 1% Triton/PBS for five minutes, washed with a hundred mM glycine/PBS for 15 minutes, blocked with 1% BSA/PBS for thirty minutes, and incubated together with the pri mary antibody overnight at four C. Samples have been subse quently washed three times for 10 minutes each and every with 1% BSA/PBS and stained together with the ideal fluorescence la beled secondary antibodies for 1 hr at space temperature.