Journal: Science Advances

Article Title: A methylation-phosphorylation switch determines Plk1 kinase activity and function in DNA damage repair

doi: 10.1126/sciadv.aau7566

Figure Lengend Snippet: ( A ) Asynchronous cells stably expressing Flag-Plk1 were treated with VP-16 (40 μM) or CPT (2 μM) for 1 hour. Plk1 were immunoprecipitated using an α-Flag antibody, and samples were probed with the indicated antibodies. ( B ) Inhibition of ATR pathway by using the ATR inhibitor VE-821 (10 μM) resulted in premature mitotic entry. Synchronized G 2 cells were treated with VP-16 (40 μM) or CPT (2 μM) for 1 hour and an addition with or without VE-821 treatment for 2 hours. The changes of K209me1 and pT210 levels were examined by Western blot. ( C , F , and H ) The indicated cells were treated with VP-16 (40 μM for 1 hour) and allowed to release into normal medium for the indicated times and analyzed using immunofluorescent staining with an α-γH2A.X, α-RPA2, or α-RAD51 antibody, respectively. ( D ) Quantification of γH2A.X-positive cells in (C) using ImageJ. The data represent means ± SD ( n > 100 each) from three independent experiments. ( E ) The indicated cells that were treated as described in (C) were analyzed using Western blotting. ( G and I ) Quantification of RPA2 or RAD51 foci numbers in individual cells described in (F) or (H) using ImageJ. The boxes designate cells with more than 10 foci, whose percentage is indicated above each box. *** P < 0.001. ( J ) The indicated cells were treated as described in (C), the chromatin fractions were collected, and chromatin-bound RPA2 and RAD51 levels were examined using Western blotting.

Article Snippet: The following antibodies and reagents were obtained from commercial sources: The α-Plk1 (F-8) (sc-17783), α-HA (Y-11) (sc-805), and α-Wee1 (B-11) (sc-5285) antibodies were purchased from Santa Cruz Biotechnology; α-Flag (F7425-2MG) and α-Aurora A (A1231) antibodies were purchased from Sigma-Aldrich; α-G9a/EHMT2(C6H3) (3306S) and α-pCdc2 (Y15) (4539T) antibodies were purchased from Cell Signaling Technology; α-cyclin B1 (1495-1), α-phospho histone H3 pS10 (1173-1), and α-phospho Plk1 pT210 (3646-1) antibodies were purchased from Epitomics; α-BubR1 (ab172581), α-RAD51 (ab133534), and α-H3K9me2 (ab1220) antibodies were purchased from Abcam; α-eGFP (50430-2-AP), α-RPA2 (10412-1-AP), α-Myc (60003-2-Ig), and α-Actinβ (60008-1-Ig) antibodies were purchased from Proteintech; α-H3K9me2 (A2359) and mouse α-GAPDH (glyceraldehyde phosphate dehydrogenase) (AC002) antibodies were purchased from ABclonal; α-H3 (39163) antibody was purchased from Active Motif; pan-mono/dimethyl (TM602) antibody was purchased from PTM Biolabs; α-HEC1(9G3.23) (GTX70268) antibody was purchased from GeneTex; α-Plk1 K209me1 antibody was generated by ABclonal by immunizing rabbits with a K209-monomethylated peptide (DGERKK(me1)TLC) conjugated with keyhole limpet hemacyanin; secondary horseradish peroxidase–conjugated α-mouse or α-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories; the α-FLAG M2 affinity gel (A2220), thymidine, and nocodazole were purchased from Sigma-Aldrich; the α–hemagglutinin (HA) affinity gel (B23301) was purchased from BioTool; BIX-01294 was purchased from Selleck; VP-16 and VE-821 were purchased from TargetMol; and CPT was purchased from Aladin.

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Inhibition, Western Blot, Staining

Journal: eLife

Article Title: BRCA1/BRC-1 and SMC-5/6 regulate DNA repair pathway engagement during Caenorhabditis elegans meiosis

doi: 10.7554/eLife.80687

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used in this study at the given dilutions: Chicken αRAD-51 (1:1000; ), Rabbit αRAD-51 (1:5000, ), Mouse αmini-AID M214-3 (1:500, MBL International), Rat αHTP-3 (1:1000, this study), Rabbit αGFP (1:500 ).

Techniques: CRISPR, Generated, Sequencing, Software