Sc 1616

Pulverized mouse lungs were homogenized in RIPA buffer (50 mM Tris HCl, pH 7.4, 1% Triton X-100, 0.2% SDS, 1 mM EDTA) supplemented with an EDTA-free protease inhibitor (Roche) and protein extracts were quantified. Western blotting was performed using 15% SDS-polyacrylamide gels and membranes were probed with antibodies raised against β -actin (Santa Cruz, sc1616, 1:1000) and RELMα (Abcam, ab39626, 1:1000). Blots were incubated with horseradish peroxidase-linked secondary antibodies and immunoreactivity was detected by enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific) and visualized with a digital luminescent image analyzer (Image Quant LAS4000 Mini; GE).

Protein was extracted from homogenised adult mouse brain (~100 mg brain; 350 μl RIPA buffer containing protease inhibitor cocktail (Roche Diagnostics, Indiana, US)) by centrifugation. Protein yield was determined by Bradford assay (Bio-Rad Laboratories, Hercules, US; bovine serum albumin (BSA) standard). Protein (80 μg) was subjected to gel electrophoresis utilising Criterion XT 1.0 mm gels (Bio-Rad) and transferred to nitrocellulose membrane (Hybond-C Extra 0.45 μm; Amersham Biosciences, Rydalmere, Aus). Membranes blocked in 3% BSA-TBS/T (Tris buffered saline (TBS)/0.1% Tween-20 (T)) overnight (4 °C) were probed for SGTA using ‘full length SGTA’ (ProteinTech), ‘C-terminal SGTA’ (Aviva BioSciences, San Diego, US) or ‘N-terminal SGTA’ (Santa Cruz) antibodies diluted at 1:1,000 (full, C-terminal) or 1:500 (N-terminal) in 1% BSA-TBS/T (60 min, room temperature). Immunoreactivity was detected using polyclonal goat anti-rabbit (1:1,000) or rabbit anti-mouse IgG/HRP (1:500) antibodies (Dako) followed by chemiluminescent visualisation (Super Signal West Dura Extended Duration, Thermo Fisher Scientific, Scoresby, Aus) and ChemiDoc MP imaging (Bio-Rad). Reference proteins used were actin (Santa Cruz Biotechnology, sc-1616, 1:2,000) or Histone H-3 (Abcam, ab7766, 1:500).