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Figure 3 | Cell Division

Figure 3

From: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation

Figure 3

Xic2 is degraded in a PCNA-dependent manner. A. Xic2 degradation assay. 35S-labeled Xic2 was incubated in HSS with (+) or without (-) ssDNA (ΦX174) with buffer (methanol) or aphidicolin (100 ng/ul). The mean percentage of remaining Xic2 from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. B. Left top panel: Sequence alignment of the PCNA binding domain in human p21Cip1, Xenopus p27Xic1, and Xenopus p16Xic2. The arrows indicate the critical PCNA binding amino acids in p21Cip1. The rectangle indicates the PIP box sequences and the circle indicates the residue (F123) mutated to disrupt PCNA binding. Left bottom panel: Xic2 GST pull-down assay. GST, GST-Xic2 wildtype (WT), or GST-Xic2 mutants (S98A, F123A, S131A, or T58A) were bound to beads and incubated with LSS. Bound fractions were analyzed by immunoblotting with α-XCyclin E (top) or α-PCNA antibody (bottom). 20% of the input reaction is shown in the left lane (LSS 2ul). Middle panel: Xic2 degradation assay. 35S-labeled Xic2 wildtype (WT) or F123A were incubated in LSS with (+) or without (-) 10 ng/ul XSC. The mean percentage of remaining Xic2 from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. Right panel: The mean relative ubiquitinated Xic2 WT or F123A from two independent experiments. C. PCNA Depletion and Xic2 degradation. Left panel: PCNA immunoblot of HSS not depleted (NOT DEPL), control-depleted (CTRL DEPL), or PCNA-depleted (PCNA DEPL). Middle panel: 35S-labeled Xic2 in HSS that was not depleted (NOT DEPL), control-depleted (CTRL DEPL), or PCNA-depleted (PCNA DEPL) with (+) or without (-) ΦX174 (ssDNA, 10 ng/ul). Right panel: The mean percentage of Xic2 remaining from two independent experiments is shown. For all figures, the ubiquitinated Xic2 protein bands are indicated as “XIC2-(UB)n”.

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