Western blot analysis.

After transfection with miRNA mimics (48 h, 50 nM), cells were washed with ice-cold 1 × PBS and cell lysis buffer (101 Bio, Palo Alto, CA), and 1× protease cocktail inhibitor mixture (Roche, Indianapolis, IN) was added. The cells were lysed by vortexing, and the plasma membrane fraction was isolated following manufacturer’s instructions. Mouse intestinal enteroids were lysed in RIPA buffer (Sigma) after transduced with lenti-mir-423-5p or negative control. Protein concentrations were determined by using Dc protein assay kit (Bio-Rad), and the samples were frozen at −80°C until further use. To examine the level of RFVT3 protein, 40 µg of soluble membrane fraction was loaded on 4–12% premade gels (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membranes. Odyssey blocking buffer was used as a blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h. The membranes were then probed with anti-hRFVT3 [1:200 dilution; rabbit polyclonal antipeptide antibodies were raised against peptides CEAPLSHLESRYLPAHFSP and LRLFSSADFCNLHCPA (which corresponds with 202–220 and 453–469, respectively) against the hRFVT3 peptide sequence (Alfa Diagnostic, San Antonio, TX)] and anti-Na⁺/K⁺ ATPase antibodies (1:50,000 dilution; Abcam, Cambridge, MA) in Odyssey blocking buffer (LI-COR) overnight at 4°C. For detecting mRFVT3 protein expression, the blot was probed with mRFVT3 polyclonal antibodies (1:500 dilution) (GeneTex, Irvine, CA). The specificity of the antibody was established before (42). The membranes were washed three times with the wash buffer containing 1 × PBS and 0.01% Tween-20 for 5 min each. PVDF membranes were then probed with corresponding secondary antibodies (LI-COR Biosciences) in 1:25,000 dilutions for 1 h at room temperature; then bands were visualized using Odyssey application software (version 3.0) in an Odyssey Infrared imaging system (LI-COR Biosciences). To confirm specificity of anti-RFVT3 antibody, an antibody and antigenic peptide (1:100) mixture was preincubated in PBS overnight, and then the PVDF membrane was probed with the mixture overnight. Membranes were further processed as described above.