Fig. 2: Differential impact of distinct enDUBs on KCNQ1 steady-state abundance, subcellular localization, and function. | Nature Communications

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

From: Decoding polyubiquitin regulation of KV7. 1 (KCNQ1) surface expression with engineered linkage-selective deubiquitinases

Fig. 2: Differential impact of distinct enDUBs on KCNQ1 steady-state abundance, subcellular localization, and function.The alternative text for this image may have been generated using AI.

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 Representative flow cytometry CDF plots showing channel abundance (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 = 10 biological replicates; #p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons: p = 0.9970, nano vs enDUB-O1; p = 0.9136, nano vs enDUB-O4; #p < 0.0001, nano vs enDUB-Cz; *p = 0.0117, nano vs enDUB-Tr; #p < 0.0001, nano vs enDUB-U21). Data were normalized to values from the nano control group (dotted line) and presented as mean ± SEM. 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, cyan; 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 = 10 biological replicates; #p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons; p = 0.9995, nano vs enDUB-O1; **p = 0.0012, nano vs enDUB-O4; **p = 0.0041, nano vs enDUB-Cz; #p < 0.0001, nano vs enDUB-Tr; p > 0.9999, nano vs enDUB-U21). Data were normalized to values from the nano control group (dotted line) and presented as mean ± SEM. F–J Co-localization of KCNQ1-YFP with subcellular markers assessed by Pearson’s co-localization coefficient (N = 3, n > 20 for each subcellular organelle; ER #p < 0.0001, one-way ANOVA and Dunnett’s multiple comparisons test: p = 0.01125, nano vs enDUB-O1; #p < 0.0001, nano vs enDUB-O4; p = 0.4620, nano vs enDUB-Cz; *p = 0.0109, nano vs enDUB-Tr; p = 0.3397, nano vs enDUB-U21. Golgi #p < 0.0001, one-way ANOVA and Dunnett’s multiple comparisons test: p = 0.9999, nano vs enDUB-O1; *p = 0.0213, nano vs enDUB-O4; **p = 0.0080, nano vs enDUB-Cz; p = 0.4119, nano vs enDUB-Tr; p = 0.9990, nano vs enDUB-U21. EE p = 0.3250, one-way ANOVA and Dunnett’s multiple comparisons test: p = 0.9992, nano vs enDUB-O1; p = 0.9998, nano vs enDUB-O4; p = 0.9845, nano vs enDUB-Cz; p = 0.8955, nano vs enDUB-Tr; p = 0.3685, nano vs enDUB-U21. LE #p < 0.0001, one-way ANOVA and Dunnett’s multiple comparisons test: p = 0.8850, nano vs enDUB-O1; p > 0.9999, nano vs enDUB-O4; p = 0.2278, nano vs enDUB-Cz; p = 0.9927, nano vs enDUB-Tr; #p < 0.0001, nano vs enDUB-U21. Lyso #p < 0.0001, one-way ANOVA and Dunnett’s multiple comparisons test: *p = 0.0209, nano vs enDUB-O1; p = 0.0645, nano vs enDUB-O4; p = 0.4973, nano vs enDUB-Cz; p = 0.1985, nano vs enDUB-Tr; p = 0.7449, nano vs enDUB-U21). 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). Pooled data were collected across N = 3 independent experiment days and presented as mean ± SEM; #p < 0.0001, two-way ANOVA, with Tukey’s multiple comparisons: p = 0.1996, nano vs enDUB-O1; #p < 0.0001, nano vs enDUB-O4; #p < 0.0001, nano vs enDUB-Cz; #p < 0.0001, nano vs enDUB-Tr; p = 9463, nano vs enDUB-U21. Source data is provided as a Source Data file.

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