Pierce bca

The surface expression of NHE3 in IPEC-J2 cells was determined separately by cell-surface biotinylation as previously described (Murtazina et al., 2006 ). Briefly, the cells were washed with cold-PBS (150 mM NaCl and 20 mM Na₂HPO₄, pH 7.4), resuspended in PBS, incubated with 1 mg/mL Sulfo-NHS-SS-Biotin (APExBIO, USA) for 30 min at room temperature, and washed with quenching buffer containing 15 mM Glycine to scavenge the unbound biotin. The cell sedimentation was lysed in RIPA buffer (Beyotime, China) containing 100 mM protease inhibitor on ice for 1 h, and the lysates were centrifuged at 11,000 × g for 15 min to remove any excess biotin reagent and byproducts. The protein concentration in the supernatants was determined using a Pierce BCA (Beyotime, China), and partial supernatants were used for an analysis of the total protein content. The amount of streptavidin agarose was calculated with protein concentrations (1 mL streptavidin agarose can bind 6 mg protein) and reacted for 90 min at 4 °C. The samples were then washed three times with RIPA containing 100 mM protease inhibitor and centrifuged at 500 × g for 30 s to remove the unbound protein. Next, 6× protein loading buffer was added and denatured at 100 °C for 5 min. A Western blot was used to analyze the percentage of the total level of cellular NHE3 expression at the apical surface of IPEC-J2 cells.

IPEC-J2 cells were washed three times with cold-PBS and lysed in radioimmunoprecipitation assay (RIPA, 200 μL/well) buffer (Beyotime, China) containing protease inhibitors (PMSF, 100 mM). The protein concentration of the resulting lysates was determined using a Pierce BCA (Beyotime, China). After centrifugation at 11,000 × g for 15 min, the proteins in the supernatant (40 μg protein) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12 % gradient gels, and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked for 1 h in Tris-buffered saline (TBS) containing 5 % non-fat dry milk at room temperature (phosphorylated proteins were blocked in 5 % BSA), and incubated with the primary antibodies at 4 °C overnight. After washing three times with TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Sangon Biotech) at 37 °C for 1 h. The proteins were visualized using 3,3′-diaminobenzidine, and detected by enhanced chemiluminescence (ECL; Thermo Scientific) and autoradiography.