Skip to main content
. 2019 Oct 25;294(48):18324–18336. doi: 10.1074/jbc.RA119.008331

Figure 5.

Figure 5.

The KDEL receptor regulates ENaC cleavage in MDCK αβγ-ENaC and CFBE41o− epithelial cells. A, C, and E, MDCK αβγ-ENaC cells were transiently co-transfected with pSK− plasmid and either nontargeting (control siRNA) or KDEL-R1 siRNA (KDELR siRNA) or co-transfected with both KDEL-R1 siRNA– and WT ERp29–expressing plasmid (KDELR siRNA/ERp29 pcDNA4). A, immunoblot analysis confirming decreased expression of KDEL-R and/or overexpression of ERp29 in whole-cell lysates (immunoblots for GAPDH and nucleolin serve as loading controls). C and E, depletion of the KDEL-R decreased baseline ENaC-mediated Isc (control, KDEL-R1 siRNA n = 24 baseline control versus KDEL-R siRNA p = 0.0003, trypsin control versus KDEL-R siRNA p = 0.0234 by ANOVA); however, overexpression of ERp29 did not rescue the decrease in baseline ENaC-medicated Isc (KDEL-R1/WT ERp29 n = 15, p = ns versus KDEL-R siRNA by ANOVA). Boldface points in C denote the representative Isc traces from E. B, D, and F, CFBE41o− cells were transfected with nontargeting (control) or KDEL-R1 siRNA. B, immunoblot analysis confirming depletion of KDEL-R expression. D and F, Isc measurements of KDEL-R–depleted CFBE41o− epithelial cells demonstrating decreased baseline ENaC-mediated Isc (n = 9, p = 0.0007). Boldface points in F denote the representative Isc traces from D. Shown are a representative surface biotinylation experiment (G) and densitometric quantification (H) of the relative expression of the β-ENaC subunits at the surface of transfected MDCK αβγ-ENaC, demonstrating that depletion of the KDEL-R does not alter β-ENaC surface expression (n = 3 independent experiments, p = ns by Wilcoxon signed-rank test). Error bars, S.D.