Figure 3.
During interphase, ubiquitination of PC4 at K68 by TRIM28 enhances the interaction with and stabilization of CCND1 mRNA. (A) Western blot showing the interaction between PC4 and TRIM28 at different cell cycle phases in Huh7 cells. (B) Western blot showing the ubiquitination level of PC4-Flag in Huh7 cells transfected with Flag-tagged PC4, HA-tagged Ub, and increasing amounts of Myc-TRIM28 as indicated. (C) Western blot showing the ubiquitination level of GST-PC4 in vitro ubiquitylation assay. The recombinant proteins GST-tagged PC4, TRIM28 along with their required cofactors were incubated at 30°C for 1 h. Blots were probed with the indicated antibodies. (D) Western blot showing the ubiquitination level of PC4-Flag in Huh7 cells transfected with Flag-tagged PC4 and Myc-tagged TRIM28 together with HA-tagged wild-type ubiquitin or K48-linked ubiquitin or K63-linked ubiquitin. IB, immunoblot. (E) Western blot showing the effect of TRIM28 on PC4 ubiquitination in Huh7 cells transduced with control or TRIM28 siRNA. (F) Western blot showing the ubiquitination levels of GST-PC4 and GST-PC4K68R in vitro ubiquitylation assay. Recombinant proteins (GST-PC4 and GST-PC4K68) were incubated with the E1, E2, and E3 for 1 h. Blots were probed with the indicated antibodies. (G) Western blot showing the ubiquitination levels of Flag-PC4 and Flag-PC4K68R in Huh7 cells transfected with Myc-tagged TRIM28, HA-tagged Ub. Ub, ubiquitin. (H) RIP-qPCR and Western blot showing the association of indicated Flag-tagged PC4 variants and CCND1 mRNA in Huh7 cells. Data are generated from n = 3 biological replicates. (I) RIP-qPCR showing the effect of TRIM28 on the association of PC4 and CCND1 mRNA. Data were generated from n = 3 biological replicates. (J) In vitro EMSA analysis showing the interaction between indicated GST-tagged recombinant PC4 variants and CCND1 5′UTR. The Ub-reaction products were incubated with CCND1 5′UTR mRNA, followed by EMSA. Data are generated from n = 3 biological replicates. (K) RIP-qPCR showing the association of indicated Flag-tagged PC4 variants and CCND1 mRNA at different cell cycle phases. Data are generated from n = 3 biological replicates. (L) Heatmap showing CCND1 mRNA stability upon ActD treatment in PC4-depleted Huh7 cells stably expressing indicated Flag-tagged PC4 variants and Myc-tagged TRIM28 at different cell cycle phases. The color bar represents the level of mRNA expression. Data are generated from n = 3 biological replicates. All quantifications are shown as mean ± SD (one-way ANOVA test); error bars represent SD. *P < 0.05, ***P < 0.001, ****P < 0.0001. A–G are representative of three independent experiments. Source data are available for this figure: SourceData F3.

During interphase, ubiquitination of PC4 at K68 by TRIM28 enhances the interaction with and stabilization of CCND1 mRNA. (A) Western blot showing the interaction between PC4 and TRIM28 at different cell cycle phases in Huh7 cells. (B) Western blot showing the ubiquitination level of PC4-Flag in Huh7 cells transfected with Flag-tagged PC4, HA-tagged Ub, and increasing amounts of Myc-TRIM28 as indicated. (C) Western blot showing the ubiquitination level of GST-PC4 in vitro ubiquitylation assay. The recombinant proteins GST-tagged PC4, TRIM28 along with their required cofactors were incubated at 30°C for 1 h. Blots were probed with the indicated antibodies. (D) Western blot showing the ubiquitination level of PC4-Flag in Huh7 cells transfected with Flag-tagged PC4 and Myc-tagged TRIM28 together with HA-tagged wild-type ubiquitin or K48-linked ubiquitin or K63-linked ubiquitin. IB, immunoblot. (E) Western blot showing the effect of TRIM28 on PC4 ubiquitination in Huh7 cells transduced with control or TRIM28 siRNA. (F) Western blot showing the ubiquitination levels of GST-PC4 and GST-PC4K68R in vitro ubiquitylation assay. Recombinant proteins (GST-PC4 and GST-PC4K68) were incubated with the E1, E2, and E3 for 1 h. Blots were probed with the indicated antibodies. (G) Western blot showing the ubiquitination levels of Flag-PC4 and Flag-PC4K68R in Huh7 cells transfected with Myc-tagged TRIM28, HA-tagged Ub. Ub, ubiquitin. (H) RIP-qPCR and Western blot showing the association of indicated Flag-tagged PC4 variants and CCND1 mRNA in Huh7 cells. Data are generated from n = 3 biological replicates. (I) RIP-qPCR showing the effect of TRIM28 on the association of PC4 and CCND1 mRNA. Data were generated from n = 3 biological replicates. (J) In vitro EMSA analysis showing the interaction between indicated GST-tagged recombinant PC4 variants and CCND1 5′UTR. The Ub-reaction products were incubated with CCND1 5′UTR mRNA, followed by EMSA. Data are generated from n = 3 biological replicates. (K) RIP-qPCR showing the association of indicated Flag-tagged PC4 variants and CCND1 mRNA at different cell cycle phases. Data are generated from n = 3 biological replicates. (L) Heatmap showing CCND1 mRNA stability upon ActD treatment in PC4-depleted Huh7 cells stably expressing indicated Flag-tagged PC4 variants and Myc-tagged TRIM28 at different cell cycle phases. The color bar represents the level of mRNA expression. Data are generated from n = 3 biological replicates. All quantifications are shown as mean ± SD (one-way ANOVA test); error bars represent SD. *P < 0.05, ***P < 0.001, ****P < 0.0001. A–G are representative of three independent experiments. Source data are available for this figure: SourceData F3.

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