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[Preprint]. 2024 Sep 17:2024.09.17.613539. [Version 1] doi: 10.1101/2024.09.17.613539

Figure 5: Differential impact of distinct linkage-selective enDUBs on reversing KCNQ1 functional downregulation by NEDD4–2.

Figure 5:

(A) Experimental design schematic; BBS-KCNQ1-YFP is co-expressed with NEDD4–2 and either nano (control) or an enDUB in HEK293 cells. (B) Left, Western blot of KCNQ1-YFP pulldowns from cells expressing KCNQ1-YFP + NEDD4–2 and either nano or the indicated enDUB, probed with anti-KCNQ1 antibody. Right, same blot stripped and probed with anti-ubiquitin. (C) Relative KCNQ1 ubiquitination computed by the ratio of anti-ubiquitin/anti-KCNQ1 signal intensities; (n=3); *p<0.05, one-way ANOVA with Dunnett’s multiple comparisons. (D) Flow cytometry representative CDF plots showing total channel expression (YFP fluorescence) in cells expressing BBS-KCNQ1-YFP + NEDD4–2 and either nano or an enDUB. (E) Mean KCNQ1 expression analyzed from YFP- and CFP- positive cells. Data were normalized to values from the nano control group (dotted line) (n>5,000 cells per experiment; N=3; ***p<0.001 and **p<0.01, one-way ANOVA with Dunnett’s multiple comparisons). (F) Representative CDF plots showing surface channels (BTX-647 fluorescence) in cells expressing BBS-KCNQ1-YFP + NEDD4–2 and either nano or an enDUB. (G) Mean BBS-KCNQ1-YFP surface expression analyzed from YFP- and CFP- positive cells. Data were normalized to values from the nano control group (dotted line) (n>5,000 cells per experiment; N=3; ***p<0.001 and #p<0.0001, one-way ANOVA with Dunnett’s multiple comparisons). (H) Exemplar KCNQ1 + KCNE1 currents from whole-cell patch clamp measurements in CHO cells. (I) Population I-V curves for nano (control; red, n=18), enDUB-O1 (turquoise, n=9), enDUB-O4 (green, n=9), enDUB-Cz (pink, n=10), enDUB-Tr (orange, n=10) and enDUB-U21 (blue, n=8). #p<0.0001, two-way ANOVA, with Tukey’s multiple comparisons.