Anti aqp1

The proteins were extracted with RIPA lysis buffer and PMSF; then, the protein quantification was conducted with a BCA protein quantification kit. Processed protein samples were collected for 8–10% SDS-PAGE electrophoresis and transferred on PVDF membranes. The membranes were blocked with 1× TBST containing 5% skimmed milk powder for 1 h at room temperature. After membrane washing, the membranes were incubated with the primary antibodies listed below at a temperature of 4 °C overnight: anti-TLR4 (1:1000, Protein-tech, Wuhan, China), anti-NF-κB (1:1000, Protein-tech, Wuhan, China), anti-MyD88 (1:1000, Protein-tech, Wuhan, China), anti-AQP1 (1:1000, Protein-tech, Wuhan, China), anti-HO-1 (1:1000, CST, Boston, MA, USA), anti-Nrf2 (1:1000, Protein-tech, Wuhan, China), and anti-β-actin (1:1000, Affinity, Changzhou, China). The membranes were then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit/mouse antibodies (1:3000, protein-tech, Wuhan, China) for 1 h at 37 °C, and the membranes were rinsed 6 times with 1× TBST to remove any remaining antibodies (5 min every time). The images were captured using an Automatic Electrophoresis Gel Imaging System (Tanon 5200 Multi, Shanghai, China).

For immunofluorescence staining, cultured TSPCs were fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were blocked with 10% normal serum blocking solution (3% bovine serum albumin and 0.1% Triton X-100 and 0.05% Tween-20) for 2 h at room temperature. After being washed, cells were incubated overnight at 4 °C with anti-AQP1 (Proteintech) and p16^INK4A (Abcam), followed by a mixture of Alexa Fluor 594-conjugated secondary antibodies (Molecular Probes) was incubated 2 h at room temperature. Immunofluorescence was visualized with a Nikon Ts2R fluorescence microscope (×20 or ×40 objectives). For EdU detection, the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 647 was used according to the manufacturer’s protocol (Beyotime Biotechnology). Immunofluorescence was visualized with an Olympus FV1000 confocal microscope (×40 objectives).

Localization of the AQP1, AQP3, AQP4, epithelial sodium channel (ENaC) and Na⁺-K⁺-ATPase proteins was evaluated in fixed gastrointestinal tissues of L. yarkandensis and O. cuniculus by immunocytochemistry. Immunocytochemical studies were performed in Paraffin-embedded gastrointestinal tissue, previously fixed in 4% paraformaldehyde. The experimental steps of immunohistochemistry have been described elsewhere^{61 (link)}. The primary antibodies used were as follows: anti-AQP1, anti-AQP3, and anti-AQP4 (diluted to 4.0 μg/ml; Proteintech) and anti-ENaC and anti-Na⁺-K⁺-ATPase (diluted to 3.0 μg/ml; Proteintech). The acquired images were analysed in IpWin32 software^{61 (link)}.