Journal: bioRxiv

Article Title: UFD1 Recognition of Initiator and Proximal Ubiquitin Drives p97-Mediated Substrate Unfolding enhanced by FAF1, FAF2, and UBXD7

doi: 10.64898/2026.03.13.711538

Figure Lengend Snippet: a, FACS-based analysis of the pexophagy flux. Representative FACS data (mKeima-SKL 561/488 ratio) for WT, FAF2 KO, or FAF2 KO stably expressing the indicated 3HA-FAF2 mutants, with the percentage of pexophagy-positive cells indicated. b, Quantitative analysis of the pexophagy flux for cells from a. Data represent the mean percentage of pexophagy-positive cells from three independent experiments. Error bars represent mean ±SEM. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001; ns. not significant. c , Cycloheximide (CHX) chase assay in HEK293T cells transfected with the indicated siRNAs for 72 h and treated with CHX for 0–6 h. Proteins were detected by western blotting using the indicated antibodies. d, CHX chase assay in HEK293T cells depleted of FAF1 and UBXD7 by siRNA (72 h) and rescued with HA-tagged WT, QY/AA (Q511A/Y515A), or ΔUBX mutant FAF1 (48 h). Cells were treated with CHX for 0–3 h. Proteins were analyzed by western blotting using the indicated antibodies. Graphs on the right show quantification of protein levels normalized to the signal at 0 h, defined as 100% (p21 or TXNIP; n = 2 biological replicates).

Article Snippet: The following antibodies were used: p21 (Cell Signaling Technologies, #2947, 1:1000), p27 (Cell Signaling Technologies, #3686, 1:1000), TXNIP (Cell Signaling Technologies, #14715, 1:1000), HIF-1α (Cell Signaling Technologies, #14179, 1:1000), FAF1 (Cell Signaling Technologies, #4932, 1:1000), UBXD7 (Thermo Fisher Scientific, #PA5-71280, 1:500), HA (Cell Signaling Technologies, #3724, 1:1000), and CDT1 (Cell Signaling Technologies, #8064, 1:1000).

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Mutagenesis

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) Western blot analysis of Myc, Skp2, and p27 expression in human mesenchymal stem cells (hMSCs) and Ewing sarcoma cells (SKES1) treated with specific siRNAs or negative control siRNA. GAPDH was used as a loading control. The inclusion of hMSCs as a non-malignant comparator clarifies disease-specific expression differences. There were decreases in each mRNA by Myc-siRNA (20 nM) and (B) Skp2-siRNA (20 nM) (B) . The expression of Myc protein was analyzed by (C) immunoblotting and (D) densitometric quantification. The effect of Skp2-siRNA on Skp2 protein expression was analyzed by (E) immunoblotting and (F) quantified accordingly.

Article Snippet: After centrifugation, supernatants were incubated with anti-p27 antibody (3686, CST) at 4 °C overnight, followed by Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) for 2 h. After washing, immunocomplexes were analyzed by SDS-PAGE and immunoblotted with anti-Ubiquitin antibody (3936, CST).

Techniques: Western Blot, Expressing, Negative Control, Control

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: Changes in the (A) phosphorylation levels of p27 and Rb protein, and (B) protein levels of Myc, CCNE, CCNA, and CDK2 by the CCNE/CDK2 complex were observed. (C) The effects of the CCNE/CDK2 complex on Rb and E2F1 binding were investigated using immunoprecipitation. The CCNE/CDK2 complex inhibited the binding of Rb to E2F1.

Article Snippet: After centrifugation, supernatants were incubated with anti-p27 antibody (3686, CST) at 4 °C overnight, followed by Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) for 2 h. After washing, immunocomplexes were analyzed by SDS-PAGE and immunoblotted with anti-Ubiquitin antibody (3936, CST).

Techniques: Phospho-proteomics, Binding Assay, Immunoprecipitation

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) The expression of ubiquitin ligase complex components was compared between Myc-overexpressing and siRNA knockdown groups. (B) Changes in p27 ubiquitination were also compared between these groups.

Article Snippet: After centrifugation, supernatants were incubated with anti-p27 antibody (3686, CST) at 4 °C overnight, followed by Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) for 2 h. After washing, immunocomplexes were analyzed by SDS-PAGE and immunoblotted with anti-Ubiquitin antibody (3936, CST).

Techniques: Expressing, Ubiquitin Proteomics, Knockdown

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) Tumor growth was compared across groups after tumor tissues were transplanted subcutaneously into mice. (B) The expression of p27 and CCNE in mouse tumor tissues was analyzed by immunohistochemistry. Original magnification, × 400; Scale bars: 50 μm. (C) The proportion of immunohistochemically positive cells was quantified. Data represent mean ± SEM of three independent experiments. **, p < 0.01 versus the related control groups. (D) A schematic summary of the study was created, demonstrating that Myc enhances the expression of AP4 and Skp2, ultimately leading to p27 ubiquitination.

Article Snippet: After centrifugation, supernatants were incubated with anti-p27 antibody (3686, CST) at 4 °C overnight, followed by Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) for 2 h. After washing, immunocomplexes were analyzed by SDS-PAGE and immunoblotted with anti-Ubiquitin antibody (3936, CST).

Techniques: Expressing, Immunohistochemistry, Control, Ubiquitin Proteomics

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) Western blot analysis of Myc, Skp2, and p27 expression in human mesenchymal stem cells (hMSCs) and Ewing sarcoma cells (SKES1) treated with specific siRNAs or negative control siRNA. GAPDH was used as a loading control. The inclusion of hMSCs as a non-malignant comparator clarifies disease-specific expression differences. There were decreases in each mRNA by Myc-siRNA (20 nM) and (B) Skp2-siRNA (20 nM) (B) . The expression of Myc protein was analyzed by (C) immunoblotting and (D) densitometric quantification. The effect of Skp2-siRNA on Skp2 protein expression was analyzed by (E) immunoblotting and (F) quantified accordingly.

Article Snippet: Formalin-fixed, paraffin-embedded tissues were sectioned (5 μm), subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0), and stained with primary antibodies against p27 (#3686, CST) and CCNE (Abcam, #ab74276).

Techniques: Western Blot, Expressing, Negative Control, Control

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: Changes in the (A) phosphorylation levels of p27 and Rb protein, and (B) protein levels of Myc, CCNE, CCNA, and CDK2 by the CCNE/CDK2 complex were observed. (C) The effects of the CCNE/CDK2 complex on Rb and E2F1 binding were investigated using immunoprecipitation. The CCNE/CDK2 complex inhibited the binding of Rb to E2F1.

Article Snippet: Formalin-fixed, paraffin-embedded tissues were sectioned (5 μm), subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0), and stained with primary antibodies against p27 (#3686, CST) and CCNE (Abcam, #ab74276).

Techniques: Phospho-proteomics, Binding Assay, Immunoprecipitation

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) The expression of ubiquitin ligase complex components was compared between Myc-overexpressing and siRNA knockdown groups. (B) Changes in p27 ubiquitination were also compared between these groups.

Article Snippet: Formalin-fixed, paraffin-embedded tissues were sectioned (5 μm), subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0), and stained with primary antibodies against p27 (#3686, CST) and CCNE (Abcam, #ab74276).

Techniques: Expressing, Ubiquitin Proteomics, Knockdown

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) Tumor growth was compared across groups after tumor tissues were transplanted subcutaneously into mice. (B) The expression of p27 and CCNE in mouse tumor tissues was analyzed by immunohistochemistry. Original magnification, × 400; Scale bars: 50 μm. (C) The proportion of immunohistochemically positive cells was quantified. Data represent mean ± SEM of three independent experiments. **, p < 0.01 versus the related control groups. (D) A schematic summary of the study was created, demonstrating that Myc enhances the expression of AP4 and Skp2, ultimately leading to p27 ubiquitination.

Article Snippet: Formalin-fixed, paraffin-embedded tissues were sectioned (5 μm), subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0), and stained with primary antibodies against p27 (#3686, CST) and CCNE (Abcam, #ab74276).

Techniques: Expressing, Immunohistochemistry, Control, Ubiquitin Proteomics