Membrane protein extraction kit

Protein expression from podocytes or isolated glomeruli was measured by immunoblotting as previously described.⁷ The Membrane Protein Extraction Kit (Sangon Biotech) was used to extract membrane proteins according to the manufacturer's protocol. Briefly, samples were separated by SDS‐PAGE and then transferred to PVDF membranes (Millipore), and the membranes were blocked for 1 h at room temperature. The membranes were then incubated overnight at 4°C with a primary antibody. Next, the membranes were washed 3 times and labelled with a secondary antibody for 1 h in the dark. Protein band intensity was visualised by an LI‐COR Odyssey Infrared Imaging System. Quantitative analysis of protein bands was performed using Image J.

Membrane proteins from the intestinal epithelial cells of the shrimp were extracted according to the instructions of the Membrane Protein Extraction Kit (Sangon Biotech Co., Ltd. China). The shrimp intestines were picked out with sterile forceps; the foregut and midgut were intercepted, and the intestinal contents were thoroughly rinsed with a sterile precooled PBS buffer. About 1 mL of an extraction buffer was added to the above tissues and homogenized with a glass homogenizer for 30–50 times. After 10 min on ice, the tissues were centrifuged at 4°C at 14,000 g for 10 min. Then the supernatant was transferred to a new centrifuge tube and placed in a water bath at 37°C for 10 min. The samples were centrifuged at 13,000 g for 5 min at room temperature and then divided into upper and lower layers (containing membrane protein). The bottom layer was removed, 500 μL of cold sterilizing water was added, and placed in an ice bath for 5 min, and at 37°C water bath for 10 min. The samples were centrifuged at 13,000 g for 5 min, and the membrane proteins were collected from the underlying aqueous phase and were saved at −80°C for later use.

The protein from tissue or cell samples was electrophoresed on SDS-PAGE, transferred to PVDF membranes (Millipore Co., Ltd.), and blocked at room temperature for 2 h. The blots were incubated with primary antibodies at 4°C overnight, washed with PBS three times, and then incubated with the secondary antibody at room temperature for 2 h or at 4°C overnight. The immunoblots were detected by ECL Western blot detection system. To determine the membrane PKCε, membrane protein was prepared with a membrane protein extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions.

The expression of ACE, AT1R, ACE2, MasR, NLRP3, ASC, pro-caspase-1, cleaved caspase-1, Na/K-ATPase and β-actin in lung tissues and Raw264.7 cells were detected by Western blotting. Briefly, RIPA lysate (CWBIO, Beijing, China) was used to extract the whole proteins in tissues and cells. Membrane Protein Extraction Kit (Sangon Biotech, Shanghai, China) was used for extraction of membrane protein. The protein analyses were carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferring membrane, and antibody incubation. Image J software (IPP, TX, USA) was used for semi-quantitative analysis. ACE (1:1000, cat. no. K003493M, Solarbio, China), AT1R (1:5000, cat. no. ab124734, Abcam, USA), ACE2 (1:5000, cat. no. ab108252, Abcam, USA), MasR (1:1000, cat. no. ab200685, Abcam, USA), NLRP3 (1:1000, cat. no. K008087P, Solarbio, China), ASC (1:1000, cat. no. #67824, Cell Signalling Technology, USA), pro-caspase-1 (1:1000, cat. no. ab179515, Abcam, USA), cleaved caspase-1 (1:1000, cat. no. #89332, Cell Signalling Technology, USA), Na/K-ATPase (1:1000, cat. no. #3010, Cell Signalling Technology, USA), β-actin (1:2000, cat. no. K101527P, Solarbio, China), and Goat Anti-rabbit IgG/HRP antibody (1:5000, cat. no. SE134, Solarbio, China).

T. reesei strains were cultured in MM medium with 10 g/L glucose and 2 g/L tryptone at 28 °C for 18 h. Mycelia were collected through filtration, the membrane protein extraction was applied using a Membrane Protein Extraction Kit (C500049, Sangon Biotech, Shang Hai, China). The membrane protein is collected in lower organic phase, and followed by precipitation with acetone. The pellet was collected through centrifugation and residual acetone was removed through volatilization on ice. The membrane protein was dissolved in 1 × SDS loading buffer and boiled for 5 min before SDS–PAGE. Western blot was carried out using a 0.45 μm PVDF membrane with standard protocol, the detection of Tre77517GFP was using Anti-GFP mouse monoclonal antibody (D191040, Sangon Biotech, Shang Hai, China) with 1:2000 dilution.