Fig. 3: Bispecific aptamer-based ZATACs induce NCL degradation in a ZYG11B- and ubiquitin proteasome-dependent manner. | Nature Communications

Fig. 3: Bispecific aptamer-based ZATACs induce NCL degradation in a ZYG11B- and ubiquitin proteasome-dependent manner.

From: Identification of a non-inhibitory aptameric ligand to CRL2ZYG11B E3 ligase for targeted protein degradation

Fig. 3

a Schematic of NCL-ZATACs-mediated degradation of NCL. b, c Microscale thermophoresis (MST) to determine the binding affinity of GFP-NCL to NCL-ZATAC#20 (b) and to the NCL-ZATAC#20/ZYG11B complex (c). d Streptavidin pulldown assays to determine the interaction between NCL and ZYG11B proteins mediated by NCL-ZATAC#20 in GFP-NCL overexpressing HEK293T cells. For Biotin-Scramble ctrl, only NCL-targeted aptamer (AS1411) is scrambled, and the Apt#Z6 remains unchanged. e Co-immunoprecipitation assays to assess the interaction between endogenous NCL and ZYG11B in the presence or absence of NCL-ZATAC#20 (500 nM) in MG132-treated (20 µM) MCF-7 cells. f–h Immunoblotting analysis of MCF-7 cells treated with the specified concentrations of Scr-ZATAC#20 (f) or NCL-ZATAC#20 for 24 h (g). The remaining NCL was quantified (h). i Immunoblotting analysis of NCL levels in MCF-7 cells treated with NCL-ZATAC#20 (500 nM) for the specified time points. j, k Immunoblotting analysis of NCL levels in MCF-7 cells treated with NCL-ZATAC#20 and cycloheximide (CHX) for specified time points (j). The remaining NCL was quantified (k). l Fluorescence images of GFP-NCL-expressing HEK293T cells treated with NCL-ZATAC#20 in the presence or absence of MG132 (20 µM). Scale bar: 100 µm. m MCF-7 cells were transfected with His-Myc-Ub plasmids, followed by treating with NCL-ZATAC#20 and MG132 (20 µM) for 5 h. Cell lysates were subjected to immunoprecipitations. n Immunoblotting analysis of MCF-7 cells treated with NCL-ZATAC#20 (500 nM) in the presence or absence of MG132 (20 µM) for 24 h. o Immunoblotting analysis of MCF-7 cells transfected with ZYG11B-targeted siRNAs, followed by treating with NCL-ZATAC#20 (500 nM) for 24 h. p Immunoblotting analysis of MCF-7 cells treated with NCL-ZATAC#20 (500 nM) and varying concentrations of Apt#Z6. Figure 3a–c were created with BioRender.com. The averages of n = 3 (b, c, h, k) biologically independent samples are shown. Data are shown as the mean ± SD. Statistical significance in (k) was assessed using the t-tests (and nonparametric tests). The data shown in (d–g, i, j, l–o) are representative of three independent experiments.

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