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

Figure 2: Differential impact of distinct enDUBs on KCNQ1 steady-state expression, subcellular localization, and function.

Figure 2:

(A) Experimental design schematic; BBS-KCNQ1-YFP is co-expressed with either GFP/YFP targeting nanobody (nano, control) or an enDUB in HEK293 cells. (B) Flow cytometry representative CDF plots showing total (YFP) fluorescence in cells expressing BBS-Q1-YFP and either nano alone (black) or each of the enDUBs (enDUB-O1, cyan; enDUB-O4, green; enDUB-Cz, magenta; enDUB-Tr, orange; enDUB-U21, blue). (C) Quantification of flow cytometry experiments for total KCNQ1-YFP expression analyzed from YFP- and CFP- positive cells (n>5,000 cells per experiment, N=10 independent experiments; #p<0.0001, *p<0.05; one-way ANOVA with Dunnett’s multiple comparisons). Data were normalized to values from the nano control group (dotted line). (D) Representative CDF plots showing surface fluorescence (BTX-647) in cells expressing BBS-KCNQ1-YFP and either nano alone (black) or an enDUB (enDUB-O1, cyane; enDUB-O4, green; enDUB-Cz, magenta; enDUB-Tr, orange; enDUB-U21, blue). (E) Quantification of flow cytometry experiments for KCNQ1-YFP surface expression analyzed from YFP- and CFP- positive cells (n>5,000 cells per experiment, N=10 independent experiments; #p<0.0001, **p<0.01; one-way ANOVA with Dunnett’s multiple comparisons). Data were normalized to values from the nano control group (dotted line). (F-J) Co-localization of KCNQ1-YFP with subcellular markers assessed by Pearson’s co-localization coefficient (N=2, n>8 for each subcellular organelle; #p<0.0001, *p<0.05, one-way ANOVA and Dunnett’s multiple comparisons test). (K) Exemplar KCNQ1 + KCNE1 current traces from whole-cell patch clamp measurements in CHO cells. (L) Population I-V curves for nano control (black, n=25), enDUB-O1 (cyan, n=9), enDUB-O4 (green, n=9), enDUB-Cz (magenta, n=16), enDUB-Tr (orange, n=16) and enDUB-U21 (blue, n=8). #p<0.0001, two-way ANOVA, with Tukey’s multiple comparisons. Here and throughout, data was pooled from n cells tested across three or more independent experimental days.