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

Figure 2.

Figure 2.

ERp29's KEEL ER retention motif is determinant in regulating ENaC cleavage in MDCK cells. A, MDCK αβγ-ENaC cells were transfected with control plasmid (pSK−), or plasmids expressing either KDEL ERp29 or ΔKEEL Erp29. ERp29 or the individual ENaC subunits were detected by immunoblot analysis of whole-cell lysate proteins using anti-ERp29, anti-HA (α-ENaC), anti-V5 (β-ENaC), and anti-γ-ENaC. B, densitometric quantification of the relative expression of the ERp29 and the ENaC subunits for n = 3–4 independent experiments (p = 0.0469 ERp29-KDEL versus control, p = 0.0190 ERp29-ΔKEEL versus control by ANOVA). C and D, MDCK αβγ-ENaC cells transfected with control plasmid (pSK−) or plasmids expressing either KDEL ERp29 or ΔKEEL Erp29 were grown as polarized monolayers and mounted in Ussing chambers for Isc measurements. C, representative Isc experiment. D, summary data for amiloride-sensitive Isc measurements normalized to control amiloride-sensitive baseline Isc (individual data points in gray and mean ± S.D. (error bars), representative data from C in black), demonstrating decreased baseline and increased trypsin response ENaC-mediated Isc versus control in ΔKEEL ERp29–transfected cells (n = 11; p = 0.0095 ΔKEEL versus control for baseline, p = 0.0320 ΔKEEL versus control for trypsin by ANOVA). For KDEL ERp29 versus control (n = 11), all p values were ns. Boldface points in D denote the representative Isc traces from C.