Chemiluminescent detection system

Randomized frozen lung samples from 3 individual mice from different groups were homogenized, and the lysates were subjected to gel electrophoresis and immunoblotting. Cytoplasmic extracts and nuclear extracts were performed by using Buffer A (10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl₂, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF), Buffer B (500 µl buffer A containing 0.5% NP-40), and Buffer C (50 mM HEPES, PH7.8, 50 mM KCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 20 µl glycerol). Briefly, lung tissues were homogenized with Buffer A. Following centrifugation, the supernatant were collected as cytoplasmic extracts, and the pellet were washed with Buffer B then re-suspended with Buffer C. Following extraction, the nuclei were removed by centrifugation, and the supernatants were collected as the nuclear extracts. Immunoreactive proteins were visualized with a chemiluminescent detection system (PerkinElmer Life Science, Inc. MA, USA) and BioMax LightFilm (Eastman Kodak Co., New Haven, CT, USA) according to the manufacturer's instructions.

Mouse hypothalamic protein lysates (20 µg protein) were separated by 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad). Following blocking in 5% skim milk, the membranes were incubated overnight at 4 °C with primary antibodies against IFT88 (1:1000). Blots were developed using a horseradish peroxidase-linked secondary antibody (1:1000) and the chemiluminescent detection system (PerkinElmer). Bands were quantified with a densitometer (VersaDoc Multi Imaging Analyzer System; Bio-Rad) and normalized to the density of β-actin.