Anti na k atpase

Human umbilical vein endothelial cells (HUVECs) and organ tissues were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors. Lysates were separated by SDS-PAGE and transferred to PVDF membranes. Target proteins were probed with specific antibodies overnight and then incubated with secondary antibodies conjugated with chemiluminescent molecules. This was followed by detection of chemiluminescent reagents (Millipore, Burlington, MA, United States) using the Bio-Rad ChemDoc MP system (Bio-Rad, Hercules, CA, United States). The primary antibodies were anti-Gsα (Santa Cruz, Dallas, TX, United States), anti-CREB (Cell Signaling Technology, Boston, MA, United States), anti-phospho-CREB Ser133 (Cell Signaling Technology), anti-GAPDH (Proteintech, Chicago, IL, United States), anti-PLVAP (Proteintech), anti-Na/K ATPase (Proteintech).

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)}.

The KCNQ1 membrane protein expression was evaluated by western blot analysis. The membrane protein was extracted with the Pierce Cell Surface Protein Isolation Kit (Thermo scientific). First, the cells were incubated with sulfo-NHs-SS-biotin marker dissolved in PBS for 30 minutes at 4°C. The above ubiquitination reaction was terminated by adding the Quenching Solution, and the supernatant was discarded by centrifugation. The adherent cells were scraped down and washed with TBS, and the lysate was added after the centrifugation. The supernatant was collected centrifugally and mixed with the washed NeutrAvidin Agarose and continuted to be incubated at room temperature for 1 hour in a shaking table. The DTT was diluted to a final concentration of 50 mM with sample buffer and incubated for 1 hour. The membrane protein was the filtrate collected by centrifugation and analyzed by western blot. The following primary antibodies were used as follows: anti-Na/K-ATPase (Proteintech, 1:500) and anti-KCNQ1 (Alomone, 1:700).