Bullet blender 24 gold

Total RNA was isolated from 5 larvae and pooled according to genotype at indicated time points. Briefly, larvae were suspended in 200 μl of Trizol (Ambion) and frozen at -80°C for at least 24 h. Upon defrosting, sterile zirconium oxide beads were added to the samples and tissue was disrupted using the Bullet Blender 24 Gold (Next Advance) for 5 min at speed level 8. Samples were spun briefly and the supernatant was subjected to phenol-chloroform extraction. DNA contaminants were removed by DNase treatment (Promega). RNA was concentrated using RNA Clean and Concentration kit (Zymo Research). RNA quality and quantity were assessed using the Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific).

The methods employed for Western blotting have been provided in detail previously [22 (link)]. Briefly, skeletal muscle tissue (~40 mg) was stabilized in buffer with protease and phosphatase inhibitor cocktail (Bimake, USA) and homogenized twice for 4 min (10,000 Bullet Blender 24 Gold, Next Advance, USA). Protein content of lysate were quantified (Pierce BCA Protein Assay Kit, Thermo Scientific, USA) samples were equilibrated in 4 × Laemmli sample buffer with DTT and heated to 95°C for 10-min with 50 μg protein subsequently loaded into separate wells on acrylamide gels (Bio-Rad, USA) for electrophoresis and wet-transferred (120-min, 70 V; Polyvinylidene difluoride membrane, Bio-Rad, USA). Membranes were washed (3 × 5-min) in Tris-buffered saline with Tween (0.05%, TBST), then blocked at room temperature for 60 min in TBST and 5% skim milk powder. Membranes were incubated in puromycin primary antibody at 4°C overnight (1: 1000 MABE343, Merck Millipore, MA, USA) and then washed and incubated in secondary antibody. Chemiluminescent solution (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific Inc., USA) was used to quantify blots by densitometry (ChemiDoc, Bio-Rad, USA) and quantified relative to total protein abundance (Amido Black Staining Solution 2 ×, Sigma-Aldrich, USA).

Lung (right superior lobe) homogenate was processed using Bullet Blender 24 Gold (Next Advance, Troy NY USA) either in PBS or RLT lysis buffer (Qiagen, Valencia, CA, USA) for protein and RNA analysis respectively. Protein from lung homogenate supernatant was assessed using the Discovery Assay^® Mouse Cytokine Array/Chemokine Array 31-Plex (MD31; Eve Technologies Corp, Calgary, AB, Canada). Total RNA from RLT lysed lung homogenate was extracted using the RNeasy mini kit (Qiagen, Valencia, CA, USA, #74104). Following RNA integrity and quantity quality control the nCounter Elements system (NanoString Technologies, Seattle, WA, USA) was employed to quantify the expression levels of 25 mouse genes (Table S2).