High speed homogenizer

Frozen lung tissues were homogenized by a high-speed homogenizer (Wuhan Servicebio Technology Co., Ltd.) and lysed in RIPA lysis buffer (cat. no. G2002; Wuhan Servicebio Technology Co., Ltd.), and total protein concentrations were measured using a Pierce BCA protein assay kit (40 (link)–42 (link)). Next, 20 µg total proteins were separated on a 10% SDS-PAGE gel according to standard protocols and electrotransferred to PVDF membranes. To block the non-specific binding of the primary antibodies, 5% BSA was used for 1 h at room temperature. Then, the membranes were incubated with the indicated primary antibodies at 4°C overnight, followed by HRP-conjugated secondary antibodies at 1:5,000 dilution (cat. no. sc-2004 and sc-2005; Santa Cruz Biotechnology, Inc.) at room temperature for an additional 1 h. The protein bands were scanned with an electrochemiluminescence reagent and the relative band intensity was quantified using Image Lab Analyzer software (version 6.0; Bio-Rad Laboratories, Inc.).

Lung homogenates were prepared in the assay buffer by a high-speed homogenizer (Wuhan Servicebio Technology Co., Ltd.) according to the respective manufacturer's instructions to determine the activities of LDH, MPO and PKA and the levels of cAMP using commercial kits. NRF2 transcription activity was detected using the TransAM^® NRF2 kit, according to the manufacturer's instructions. Briefly, nuclear extracts were prepared from fresh lung tissues and incubated in plates coated with oligonucleotides containing an antioxidant responsive element. Subsequently, a primary antibody against NRF2 and a horseradish peroxidase (HRP)-conjugated secondary antibody were added. Then, the absorbance was read at 450 nm on a spectrophotometer with a reference wavelength of 655 nm.