Journal: Cell reports

Article Title: DCAF14 promotes stalled fork stability to maintain genome integrity

doi: 10.1016/j.celrep.2020.108669

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-DDB1 , Cell Signaling Technology , Cat#5428S; RRID:AB_10634753.

Techniques: Virus, Recombinant, Transfection, In Situ, Single Cell Gel Electrophoresis, Negative Control, Expressing, Plasmid Preparation, Software

Journal: Oncogene

Article Title: Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin.

doi: 10.1038/onc.2016.336

Figure Lengend Snippet: Figure 3. UBR5-containing EDVP E3 ligase complex interacts and regulates MOAP-1 ubiquitylation and stability. (a) Flag-MOAP-1 was transfected into 293T cells, and lysates were prepared 48 h posttransfection. Co-IP with Flag M2 agarose beads were performed and immunoblotted with antibodies as indicated. n = 3 independent experiments. (b) In vitro ubiquitylation assay was performed as Figure 2d in the presence or absence of recombinant DDB1, VprBP and Dyrk2. n = 3 independent experiments. Original uncropped image is shown in Supplementary Figure S6. (c) siCtrl or siDyrk2 was transfected into H1299 or 293T cells, and Flag-MOAP-1 was transfected into each siRNA transfectant 24 h post-siRNA transfection. Cell lysates were prepared under denaturing condition; Flag-MOAP-1 was immunoprecipitated and immunoblotted with ubiquitin antibody. Same membrane was re-blotted with Flag antibody for Flag-MOAP-1. n = 3 independent experiments. Asterisk in Dyrk2 blot indicates non-specific band. Quantification of MOAP-1 ubiquitylation is shown in Supplementary Figure S3B. (d) 293T cells transfected with siCtrl, siDyrk2 or siUBR5 were treated with CHX 48 h posttransfection. Cells were collected at the indicated times after CHX treatment, and lysates were prepared and immunoblotted as indicated (left). MOAP-1 protein level was quantified and plotted (right). The MOAP-1 abundance at 0 time point was set at 100%. n = 4 independent experiments (means ± s.e.m.). Molecular weight markers are in kDa.

Article Snippet: Antibodies Antibodies used in this study are as follows: MOAP-1 rabbit polyclonal (Cat. no. HPA000939, Sigma/Atlas antibodies, Stockholm, Sweden), UBR5 rabbit polyclonal (Cat. no. 8755, Cell Signaling Technology, Danvers, MA, USA), VprBP Oncogene (2017) 1698 – 1706 rabbit polyclonal (Cat. no. A301-887A, Bethyl Laboratories, Montgomery, TX, USA), DDB1 rabbit polyclonal (Cat. no. A300-462A, Bethyl Laboratories), DUBA rabbit polyclonal (Cat. no. A302-919A, Bethyl Laboratories), Flag M2 mouse monoclonal (Cat. no. F1804, Sigma), Flag rabbit polyclonal (Cat. no. PA1-984B, Thermo Scientific, Waltham, MA, USA), Cdh1 mouse monoclonal (DH01, Cat. no. MS-1116-P, Thermo Scientific), Cyclin B1 mouse monoclonal (GNS1, Cat. no. sc245, Santa Cruz Biotechnology, Dallas, TX, USA), β-actin mouse monoclonal (Cat. no. ab8224, Abcam, Cambridge, UK), actin rabbit polyclonal (I-19-R, Cat. no. sc1616-R, Santa Cruz Biotechnology), Dyrk2 rabbit polyclonal (Cat. no. ab37912, Abcam or Cat. no. 8143, Cell Signaling Technology), PEPCK1 rabbit monoclonal (D12F5, Cat. no. 12940, Cell Signaling Technology), ubiquitin mouse monoclonal (P4D1, Cat. no. sc-8017, Santa Cruz Biotechnology), active Bax 6A7 mouse monoclonal (Cat. no. 556467, BD Biosciences, Franklin Lakes, NJ, USA), Bax rabbit polyclonal (N-20, Cat. no. sc-493, Santa Cruz Biotechnology), Hsp70 mouse monoclonal (Cat. no. 610607, BD Biosciences).

Techniques: Transfection, Co-Immunoprecipitation Assay, In Vitro, Ubiquitin Assay, Recombinant, Immunoprecipitation, Ubiquitin Proteomics, Membrane, Molecular Weight