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. 2022 Jan 20;50(3):1517–1530. doi: 10.1093/nar/gkac011

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — TRIM21 ubiquitinated CLASPIN with K63 linkage that counteracted its K6-linkage ubiquitination. (A, B) HEK293T cells were transfected with a negative control or a TRIM21-specific siRNA for 24 h. Then, the cells were co-transfected with FLAG-CLASPIN and HA-ubiquitin K63-only (as in A) or HA-ubiquitin K6-only (as in B) together with MYC-TRIM21 or MYC-TRIM21 E3 ligase inactive mutant as indicated. After 24 h, all the cell lines were lysed and subjected to de-naturing immunoprecipitation using anti-FLAG M2 agarose. The immunoprecipitates were examined by immunoblotting with the indicated antibodies. (C, D) HEK293T cells were transfected with FLAG-CLASPIN, and HA-ubiquitin K6-only along with increasing doses of MYC-ubiquitin K63-only expressing plasmids (as in C) or HA-ubiquitin K63-only along with increasing doses of MYC-ubiquitin K6-only expressing plasmids (as in D). After 24 h, the cells were lysed and analyzed by de-naturing immunoprecipitation using anti-FLAG M2 agarose, followed by immunoblotting with the indicated antibodies. (E) HEK293T cells were co-transfected with HA-ubiquitin K63-only and full-length FLAG-CLASPIN or its deletion mutants, and then analyzed by de-naturing immunoprecipitation and immunoblotting with the indicated antibodies. (F) In vitro ubiquitination assays were performed by incubating HIS-TRIM21 or TRIM21CA with GST-CLASPIN 301–630aa in the presence of E1, E2 and HA-ubiquitin-K63 only (HA-UB(K63)) at 30°C for 1 h, followed by GST-pulldown and analysis by immunoblotting with the indicated antibodies. (G) K63-linked ubiquitination of wildtype CLASPIN and its deletion mutants was examined as described in (E). (H) HEK293T cells were co-transfected with HA-ubiquitin K6-only and full-length FLAG-CLASPIN or its deletion mutants, and analyzed by denaturing immunoprecipitation and immunoblotting with the indicated antibodies. (I) HEK293T cells were transfected with the indicated plasmids and analyzed by de-naturing immunoprecipitation and immunoblotting with the indicated antibodies. (J, K) HEK293T cells were co-transfected with MYC-ubiquitin K63-only (as in J) or MYC-ubiquitin K6-only (as in K) and wildtype FLAG-CLASPIN or its K565/580/581R mutant. The samples were analyzed by de-naturing immunoprecipitation and immunoblotting with the indicated antibodies.