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

Figure 5.

Figure 5.

TRIM21-mediated CLASPIN ubiquitination suppressed CHK1 activation. (A) HEK293T cells were transfected with a negative control or a siRNA targeting for the 3’UTR of CLASPIN (siCLASPIN) for 24 h. Then, the cells were transfected with FLAG-CLASPIN or FLAG-CLASPINΔ501–600aa as indicated and incubated for a further 24 h. Then the cells were treated with 2 mM HU or a mock treatment before the whole cell lysates were collected and analyzed by immunoblotting. Activation of CHK1 was examined by immunoblotting with an antibody specifically recognizing phosphorylated CHK1 at Ser345. The CHK1 and CLASPIN expression levels were also examined. Actin: loading control. *, non-specific signal. Statistical analysis (t-test) of signal intensity from three independent experiments was shown in the lower panel. **P < 0.01; ***P < 0.001. (B) HEK293T cells were transfected with a negative control or a CLASPIN-specific siRNA for 24 h. Then, the cells were transfected with FLAG-CLASPIN or FLAG-CLASPIN K565/580/581R (3KR) mutant as indicated, and incubated for a further 24 h. Then the cells were treated and examined as in (A). *, non-specific signal. Statistical analysis (t-test) of signal intensity from three independent experiments was shown in the lower panel. **P < 0.01; ***P < 0.001.