Image studio lite analysis software

Ten sections from the midcostal part of the diaphragm were dissolved in SDS buffer (50 mM Tris-HCl, 2%SDS, 0.1% bromophenol blue, 10% glycerol, and 2%β-mercaptoethanol, pH 8.8), heated for 2 min at 95°C and total protein was quantified using the Pierce 660 nm Protein Assembly Assay (Thermo Scientific). Equal amounts of protein were separated on SDS-polyacrylamide gels (Mini-PROTEAN 3 Cell, Bio-Rad Laboratories) at constant 120 volts for 90 min. Acrylamide concentrations were 4 and 12% (w/v) in stacking and running gels respectively, and the gel matrix included 10% glycerol. Separated proteins were transferred to poly(vinylidene difluoride) membranes (Immobilon-P, Millipore). Membranes were blocked with 3% non-fat milk powder in Tris-buffered saline containing Tween 20 for 1 h and incubated overnight at 4°C with Hsp72- (SMC-100B, StressMarq) and actin (sc-1616, Santa Cruz Biotechnology Inc.) primary antibodies and then with HRP secondary antibodies (anti-mouse, GE Healthcare, and anti-goat, Santa Cruz Biotech). Membranes were digitized with ECL 500 Western Blotting Detection Agent Kit (Amersham Biosciences) and imaging system (Odyssey, LI-COR Biosciences). Band densities were quantified with Image Studio Lite analysis software (LI-COR Biosciences) and normalized to actin.

Cells were lysed using M-PER mammalian protein extraction reagent (Thermo Scientific) containing a protease inhibitors cocktail (Roche). The lysates were centrifuged at 4°C for 20 min at 16000g and supernatants were collected. Total protein concentration was determined using Precision Red advanced protein assay reagent (ADV02, Cytoskeleton). The supernatants were incubated for 5 min at 95°C with NuPAGE^® LDS sample buffer (NP007, Life Technologies). Equal amounts of protein were loaded and resolved using precast gels from Bio-rad (Mini PROTEAN TGX Gel, Cat number 456-9034) and electro-transferred to nitrocellulose membranes (Hybond, GE Healthcare). The blots were blocked with 5% milk powder and incubated with primary antibody according to the manufacturer’s protocol. Secondary antibody conjugated with horseradish peroxidase (HRP) was used for detection using ECL plus western blotting detection reagent (GE Healthcare). To confirm equal protein loading, all the immunoblots were also probed with anti GAPDH antibody. The immunoblots were scanned on the LI-COR Odyssey FC Imaging System and the signal intensity was analyzed by Image Studio™ Lite analysis software (LI-COR).