Fig 1: Protein expression of selected ion transporters and carbonic anhydrase isoforms in human duodenal enteroid monolayers. (A) Representative immunoblots showing the protein expression of selected ion transporters and carbonic anhydrase isoforms in undifferentiated (UD) and differentiated (DF) duodenal enteroid monolayers derived from 3 donors. Immunoblotting was performed using 4%–20% gradient gel or 8% gel (for DRA and CFTR). The immunoblot of DRA shows 2 bands representing 2 glycosylated forms of DRA protein, both of which were used for quantitation (see Figure 4C and D). Note that the nitrocellulose membranes were cut into 2 pieces to be blotted with multiple primary antibodies. Proteins that were blotted with rabbit polyclonal antibodies are shown in red, proteins that were blotted with mouse monoclonal antibodies are shown in green. (B) Quantitation showing a significant increase in the protein expression of CA2, DRA, NBCe1, NHE3, and a decrease in CFTR and NKCC1 after differentiation. Calculation was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. *P < .05, **P < .01, *** P < .001. n = 6–13 experiments with paired enteroid monolayers derived from 3–4 donors. (C and D) Protein expression of DRA was studied by immunoblotting using mouse monoclonal primary antibody (sc-376187; Santa Cruz, Dallas, TX). The comparison between wild type HEK cells (lacking DRA mRNA and protein) and HEK cells with transient transfection of 3xFLAG-DRA showed 2 potential DRA bands. A deglycosylation study was performed using PNGase F (New England Biolabs, Ipswich, MA) according to the manufacturer’s protocols, showing the 2 bands represent 2 glycosylated forms of DRA protein in differentiated duodenal enteroid monolayers (endogenous DRA expression) and HEK cells with transient transfection of 3xFLAG-DRA (exogenous DRA expression).