Journal: BMB Reports

Article Title: APC/C−CDH1 suppresses AROS-mediated protection against DNA damage-induced senescence by ubiquitination

doi: 10.5483/BMBRep.2025-0031

Figure Lengend Snippet: AROS is ubiquitinated and degraded by APC/C−CDH1. (A) CDH1 promotes AROS degradation. HEK293 cells were co-transfected with MyC−CDH1 or Myc–CDC20 with Flag–AROS. Cell lysates were analyzed by Western blotting with the indicated antibodies. (B) CDH1-mediated degradation of AROS was restored by treatment with the proteasome inhibitor MG132. (C) CHX chase assays. HEK293 cells were co-transfected with Flag–AROS and Myc–Empty or MyC−CDH1 for 12 h. Cells were then treated with CHX (50 μg/ml) for the indicated times, and Western blotting was performed with the indicated antibodies. Degradation of AROS was quantified using ImageJ software. Error bars represent the mean ± SD of three independent experiments (**P < 0.01, ***P < 0.001). (D) CDH1 knockdown increases AROS levels. HEK293 cells were transfected with sh–CDH1 or sh–Luciferase (Luc) as a control. After 48 h, cell lysates were subjected to Western blotting with the indicated antibodies. (E) AROS is ubiquitinated by CDH1. HEK293 cells were co-transfected under the indicated conditions for 24 h, followed by treatment with 10 μM MG132 for 12 h, then lysed, and subjected to IP using anti-Flag antibody. AROS ubiquitination was assessed by Western blotting with anti-Ub antibody. (F) In vitro ubiquitination of AROS by CDH1. In vitro -translated Flag–AROS and MyC−CDH1 were incubated with APC/C immunoprecipitated with anti-APC2 antibody using a ubiquitination kit. Ubiquitinated AROS was visualized by Western blotting with anti-poly–Ub antibody.

Article Snippet: The following antibodies were used: AROS (sc–86209; Peptron or Santa Cruz Biotechnology), APC2 (12301S, Cell Signaling Technology), CDH1 (ab3242; Abcam), CDC20 (sc13162; Santa Cruz Biotechnology), Cyclin B1 (sc245; Santa Cruz Biotechnology), polyubiquitinated conjugates (BML−PW8805−0500; Enzo Life Science), Flag M2 (F1804; Sigma–Aldrich), Myc (05−724; Merck Millipore), GFP (sc–8334; Santa Cruz Biotechnology), HA (sc7392; Santa Cruz Biotechnology), and β–actin (sc47778; Santa Cruz Biotechnology).

Techniques: Transfection, Western Blot, Software, Knockdown, Luciferase, Control, Ubiquitin Proteomics, In Vitro, Incubation, Immunoprecipitation

Journal: Science Advances

Article Title: Deubiquitinase USP8 regulates the spindle assembly checkpoint in oocytes

doi: 10.1126/sciadv.aeb2345

Figure Lengend Snippet: ( A ) Protein levels of the MCC were assessed by immunoblots in control and USP8-depleted oocytes. The blots were probed with USP8, BUBR1, CDC20, BUB3, MAD2, and Cofilin antibodies. ( B ) Localization of BUB3 at the prometaphase I stage in control, USP8-KD, and USP8-rescue oocytes. At 3 hours after GVBD, oocytes were fixed and immunostained for BUB3, CREST, and DNA (Hoechst). Scale bar, 10 μm. ( C ) The relative fluorescence intensities of BUB3 to CREST were measured in control ( n = 200, kinetochores), USP8-KD ( n = 200, kinetochores), and USP8-rescue ( n = 200, kinetochores) oocytes. The signal intensity of BUB3 was normalized with that of CREST. ( D ) Protein levels of BUB3 were assessed by immunoblots in control, USP8-KD, and USP8-rescue oocytes. ( E ) Co-IP result showing the USP8 interaction with BUB3 in oocytes. Mouse oocytes were microinjected with USP8-Flag and BUB3-HA cRNA together or USP8-Flag cRNA alone, maintained for a further 4 hours in 200 μM IBMX to allow time for translation. Target proteins were immunoprecipitated using anti-Flag beads and subjected to Western blotting with Flag and HA antibodies. Input oocyte lysates were immunoblotted with anti-HA antibody to determine the expression of BUB3. ( F ) Co-IP result showing the BUB3 interaction with USP8 in oocytes. Mouse oocytes were microinjected with BUB3-HA and USP8-Flag cRNA together or BUB3-HA cRNA alone, maintained for a further 4 hours in 200 μM IBMX to allow time for translation. Target proteins were immunoprecipitated using anti-HA beads and subjected to Western blotting with HA and Flag antibodies. Input oocyte lysates were immunoblotted with anti-Flag antibody to determine the expression of USP8. Data were presented as the mean percentage (means ± SEM) of at least three independent experiments. *** P < 0.001.

Article Snippet: The blots were further blocked in TBST containing 5% low-fat dry milk for 1 hour at room temperature and then incubated with USP8 (1:500; Abclonal, A7031), USP8 (1:500; Proteintech, 67321-1-Ig), Cyclin B1 (1:1000; Cell Signaling Technology, 4135), BUB3 (1:1000; Abcam, ab133699), BUBR1 (1:1000; Abcam, Ab28193), MAD2 (1:1000; Proteintech, 10337-1-AP), CDC20 (1:500; Proteintech, 10252-1-AP), BUB1 (1:1000; Abcam, Ab195268 ), MPS1 (1:500; Proteintech, 10381-1-AP), Flag (1:1000; Sigma-Aldrich, F3165), HA (1:1000; Sigma-Aldrich, H9658), MYC (1:1000; Cell Signaling Technology, 2278), Normal Rabbit IgG (1:1000; Cell Signaling Technology, 2729), or Cofilin (1:5000; Proteintech, 66057-1-Ig) antibodies at 4°C overnight.

Techniques: Western Blot, Control, Fluorescence, Co-Immunoprecipitation Assay, Immunoprecipitation, Expressing