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

Figure 4.

Figure 4.

TRIM21 negatively regulated the chromatin loading of CLASPIN and its interaction with TIPIN. (A) HEK293T cells were treated with 2 mM HU for the indicated times, total cell lysates were extracted and subjected to immunoprecipitation and immunoblotting with antibodies as indicated. *, non-specific signal. (B) HeLa cells were transfected with siNC or siTRIM21 for 24 h and subjected to double thymidine blocks to synchronize cells in G1/S transition followed by a release for 2 h. Transfectants were untreated or treated with 2 mM HU for 1 h before chromatin fractionation was performed. The chromatin-enriched fraction was analyzed by immunoblotting with the indicated antibodies. * Non-specific signal. Statistical analysis (t-test) of signal intensity from three independent experiments was shown in the lower panel. *P < 0.05; ***P < 0.001. (C) HEK293T cells were transfected with FLAG-CLASPIN or the FLAG-CLASPINΔ501–600 mutant. After 24 h, the cells were treated with 2 mM HU or a mock treatment for 1 h and subjected to chromatin-bound protein extraction. The protein levels of FLAG-CLASPIN or FLAG-CLASPINΔ501–600aa in the chromatin fraction and whole cell lysate were detected by immunoblotting, and relative chromatin loading was analyzed by comparing the ratio between the chromatin fraction and the whole cell lysate. (D) HEK293T cells were transfected with FLAG-CLASPIN or the FLAG-CLASPIN K565/580/581R. Then cells were treated and analyzed by immunoblotting as described in (C). (E) HEK293T cells were transfected with HA-TIPIN and FLAG-CLASPIN or its Δ501–600aa mutant. After 24 h, the cells were treated with 2 mM HU or a mock treatment for 1 h, and subjected to immunoprecipitation using anti-FLAG M2 agarose, followed by immunoblotting with the indicated antibodies. (F) HEK293T cells were transfected with HA-TIPIN and FLAG-CLASPIN or its K565/580/581R mutant. After 24 h, the cells were treated with 2 mM HU or a mock treatment for 1 h and subjected to immunoprecipitation using anti-FLAG M2 agarose, followed by immunoblotting with the indicated antibodies. (G) HEK293T cells were transfected with a negative control or different TRIM21 specific siRNAs for 24 h. Then the cells were transfected with HA-TIPIN and FLAG-CLASPIN or its Δ501–600aa mutant, and analyzed by immunoprecipitation using anti-FLAG M2 agarose followed by immunoblotting with the indicated antibodies.