anti ube2s  (Cell Signaling Technology Inc)

Journal: PLOS Biology

Article Title: The deubiquitinating enzyme Cezanne stabilizes BRCA1 by counteracting APC/C and Ube2S-dependent Lys11-linked ubiquitination

doi: 10.1371/journal.pbio.3003545

Figure Lengend Snippet: (A) Increased K11-ubiquitination of BRCA1 in Cezanne KO cells untreated or treated with IR. Flag IP was performed under denaturing condition using lysates of WT or Cezanne KO U2OS cells expressing Flag-BRCA1 in combination with or without HA-K11 Ub. Cells were either untreated or treated with IR (10 Gy, 2 h). MG132 was added 6 h before harvest of cells. (B) Cezanne knockdown leads to increased endogenous BRCA1 K11-ubiquitination. BRCA1 IP was performed under denaturing condition. U2OS cells expressing HA-K11 Ub were treated with indicated siRNAs. MG132 was added 6 h before harvest of cells. (C) Increased endogenous BRCA1 K11-ubiquitination in Cezanne-depleted cells is independent of IR. U2OS cells were transfected with HA-K11-Ub and treated with indicated siRNA. Cells were also untreated or treated with IR (10 Gy, 2 h) . MG132 was added 6 h before harvest of cells. BRCA1 IP was performed under denaturing condition. (D) Cezanne DUB activity is required for K11 ubiquitination of BRCA1. U2OS WT or Cezanne KO (Cez-KO) cells, as well as Cez-KO cells complemented with expression of Cezanne WT or DUB-inactive CH mutant, were transfected with HA-K11 Ub. MG132 was added 6 h before harvest of cells. “SE”, short exposure; “LE” long exposure. BRCA1 IP was performed under denaturing condition. (E) Overexpression of Cezanne decreases BRCA1 K11-ubiquitination. U2OS cells were transfected with Flag-BRCA1 and with or without HA-K11-Ub, GFP-tagged wild-type (WT) or CH mutant of Cezanne. MG132 was added 6 h before harvest of cells. Flag IP was performed under denaturing condition. (F) Depletion of Ube2S reduces the increased K11-ubiquitination of BRCA1 in Cezanne-deficient cells. U2OS cells were transfected with HA-K11-Ub and Flag-BRCA1, followed by transfection with indicated siRNAs. MG132 was added 6 h before harvest of cells. Flag IP was performed under denaturing condition. (G) Depletion of Ube2S restores BRCA1 protein level in Cezanne-siRNA-treated cells. Total lysates of cells treated with indicated siRNAs were examined by western blots. Relative amount of BRCA1 is measured by Image J and quantified from three independent experiments. GAPDH in (A) (B) (C) (D) (F) (G) and tubulin in (E) was used as a loading control. Western blots shown are representative of three independent experiments. The data underlying the graph shown in the figure can be found in .

Article Snippet: Antibodies used are: BRCA1 (Cat#sc-6954, Santa Cruz), Cezanne (Cat#sc-514402, Santa Cruz), Ube2S (Cat#9630, Cell Signaling), Ube2C (Cat#WH0011065M1, Sigma), HA (Cat#9661S, Cell Signaling), Flag (Cat#F7425, Sigma), GFP (Cat#A11122, Invitrogen), Cyclin B1 (Cat#sc245, Santa Cruz), Cyclin A (Cat#sc596, Santa Cruz), CDH1 (Invitrogen, Cat#34-2000), APC2 (Cell Signaling, Cat#12301), BARD1 (Proteintech, Cat#22964-1AP).

Techniques: Ubiquitin Proteomics, Expressing, Knockdown, Transfection, Activity Assay, Mutagenesis, Over Expression, Western Blot, Control

Journal: PLOS Biology

Article Title: The deubiquitinating enzyme Cezanne stabilizes BRCA1 by counteracting APC/C and Ube2S-dependent Lys11-linked ubiquitination

doi: 10.1371/journal.pbio.3003545

Figure Lengend Snippet: (A) Ube2S overexpression reduces BRCA1 protein level. Total cell lysates from U2OS cells with or without expression of GFP-Ube2S were analyzed with western blots. GAPDH was used as a loading control. Western blots shown are representative of three independent experiments. (B) Upregulation of Ube2S in U2OS cells leads to increased cellular sensitivity to olaparib detected by colony formation assay. Quantifications are shown with mean ± SD. Ordinary Two-way Anova was used for statistics. Data are representative of three independent experiments. (C) Increased drug sensitivity to olaparib in UBE2S-high breast cancer cell lines. CCLE breast cancer cell lines were stratified by Kmeans 2 separation of UBE2S expression. IC50 data were obtained from GDSC. P value was generated by Student t test. (D) Contribution of the mutational signature 3 in TCGA BRCA patients stratified by Kmeans 3 separation of Ube2S expression levels (patient numbers as depicted). P value was generated by Student t test of low and high groups. (E) Correlation of Ube2S expression to a 30-gene BRCAness signature in breast tumors. Left, heatmap of BRCAness signature genes in TCGA BRCA tumors ordered by Ube2S expression. Right, scatter plot of Ube2S expression against BRCAness signature scores. Pearson correlation coefficient is shown. (F) A proposed model for cell cycle-dependent regulation of BRCA1 protein level in G1 and S phase by the countering activities of APC/C Cdh1 /Ube2S and Cezanne in K11-linkage specific ubiquitination of BRCA1. Dysregulation of this pathway due to loss of Cezanne or upregulation of Ube2S leads to reduced BRCA1 protein level and is associated with BRCAness in tumors. The data underlying the graphs shown in the figure can be found in .

Article Snippet: Antibodies used are: BRCA1 (Cat#sc-6954, Santa Cruz), Cezanne (Cat#sc-514402, Santa Cruz), Ube2S (Cat#9630, Cell Signaling), Ube2C (Cat#WH0011065M1, Sigma), HA (Cat#9661S, Cell Signaling), Flag (Cat#F7425, Sigma), GFP (Cat#A11122, Invitrogen), Cyclin B1 (Cat#sc245, Santa Cruz), Cyclin A (Cat#sc596, Santa Cruz), CDH1 (Invitrogen, Cat#34-2000), APC2 (Cell Signaling, Cat#12301), BARD1 (Proteintech, Cat#22964-1AP).

Techniques: Over Expression, Expressing, Western Blot, Control, Colony Assay, Generated, Ubiquitin Proteomics