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Cell extracts for both knock-downs and untreated controls were prepared in RIPA buffer (Thermo) supplemented with complete protease inhibitor tablets (Roche). The total protein concentration was determined via the Lowry method (BioRad) and 10ug of total protein were separated by 10% SDS–polyacrylamide gel (Mini PROTEAN TGX, BioRad) electrophoresis. The membrane was blocked with 5% milk protein (Cell Signaling) in TBST (20 mM Tris, pH = 7,5, 150 mM NaCl, 0,1% Tween 20) and probed overnight at 4 °C with mouse monoclonal anti-CFTR antibody (sc-376683, Santa Cruz) and β-actin (A5441, Sigma-Aldrich). As secondary antibody, we used horseradish peroxidase-conjugated donkey anti-mouse IgG (A16017, Invitrogen).

Caco-2 cell lysates were collected on ice in RIPA buffer and isolated by centrifugation at 13,000 RPM for 10 min at 4 °C. Protein was quantified by Pierce BCA (Catalog#: 23225, Thermo Fisher, Waltham, MA, USA). 30 μg protein was boiled with sample buffer prior to loading on a polyacrylamide gel. SLC26A3 antibody diluted 1:1000 in TBS-T + 5% milk (Catalog#: GTX34204, GeneTex, Irvine, CA, USA), CFTR antibody diluted 1:500 in TBS-T + 5% milk (Catalog#:sc-376683, Santa Cruz Biotechnology, Dallas, TX, USA), ZO-1 antibody diluted 1:1000 in TBS-T + 5% milk (Catalog#: GTX108613, GeneTex, Irvine, CA, USA), GAPDH antibody diluted 1:2000 in TBS-T + 5% milk (Catalog#: Sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA). Goat anti-rabbit HRP antibody diluted 1:10,000 in TBS-T + 5% milk (Catalog#: PI-1000, Vector Biolabs, Malvern, PA, USA) and horse anti-mouse HRP antibody diluted 1:10,000 in TBS-T + 5% milk (Catalog#: P1-2000, vector Biolabs Malvern, PA, USA) were used to visualize westerns. Bands were visualized by ECL (Pierce ECL Western Blotting Substrate, Catalog#: 32106, Thermo Fisher,Waltham, MA, USA) with a FluorChem M Imager (ProteinSimple, San Jose, CA, USA) and quantified by AlphaView Software SA, v.3.4.0.0.

Paraffin-embedding of kidney specimens and sectioning were performed via standardized procedures. The resulting sections underwent staining with periodic acid-Schiff (G1360; Solarbio, China) and Sirius red staining (BB-44333; BestBio, China) reagents according to respective recommended protocols. Immunohistochemical staining was carried out using a Two Step Immunoassay assay kit (ZSBIO, China), as directed by the manufacturer. The antibodies used included rabbit monoclonal anti-CDX2 (YM3057; Immunoway Bio, USA), mouse monoclonal anti-CFTR (sc-376683; Santa Cruz, USA), and rabbit polyclonal anti-β-catenin (bs-1165R; Bioss, Beijing, China). Areas of positive staining were quantified by ImageJ in 6 random fields (200×) per sample, with three individuals assessed in each group.