mouse anti brca2  (R&D Systems)

Journal: Nature Communications

Article Title: Sulphostin-inspired N -phosphonopiperidones as selective covalent DPP8 and DPP9 inhibitors

doi: 10.1038/s41467-025-58493-z

Figure Lengend Snippet: a X-ray structure of 16 in complex with DPP9 refined to a final resolution of 2.49 Å, shown from two different angles. The ligand is covalently bound to the catalytically active serine S730 residue and is contoured at 1 σ with the refined 2F o -F c electron density map. Hydrogen bond interactions are depicted as dashed lines. b ABPP without (FC plotted on x-axis) or after pre-treatment with 50 µM 8, 11 or 16 (competition experiment, FC plotted on y-axis) with 10 µM 9 after click chemistry ( n = 4 biologically independent samples). Dashed lines indicate the FC ≥ 2 ( 9 /DMSO) or FC ≤ -2 (Competitor/ 9 ) threshold. Black symbols indicate identified protein groups. To identify statistically significant hits from the analysis (marked as a triangle or square), p ≤ 0.01 (two-sided Student’s t -test, permutation-based FDR with 250 randomizations and FDR = 0.05) was applied. c Addition of N -phosphono - ( S )−3-aminopiperidine-2-ones block the interaction between endogenous DPP9 and BRCA2. Quantification of PLAs showing MMC-induced DPP9-BRCA2 PLA events in HeLa wild-type cells, in the presence of 10 µM of the respective inhibitory compounds. Cells were treated with 300 nM MMC for 24 h and 10 µM of the indicated inhibitors for 1 h prior to fixation. Each dot represents the number of PLA events in a single cell. Data were analyzed by unpaired two-sided t -test comparisons (**** p < 0.0001). To visualize the three biological replicates, each biological replicate is labeled differently: circle, triangle or square. n values indicate total number of cells in each analysis group from 3 independent biological replicates in total: n = 240 for BRCA2 Ab-Ctrl., n = 237 for DPP9 Ab-Ctrl., n = 222 for + Ctrl., n = 202 for 1G244, n = 231 for 8, n = 205 for 16 . d Inhibition of DPP8/9 increases cellular sensitivity to genotoxic stress. HeLa wild-type (WT) cells were treated with 1 µM MMC and 10 µM of the respective inhibitors. Cell viability was measured after 72 h, and normalized to WT mock treated cells. DPP9 KO cells and WT cells treated with 1G244 (10 µM) were analyzed as positive controls, whereas WT cells treated with Sitagliptin were used as a negative control. Dashed lines indicate the viability of the WT + MMC cells. The graph shows the mean and error bars indicate the SEM of all individual measurements ( n = 18 (HeLa wild-type controls), 17 (HeLa DPP9 KO control) or 9 (inhibitor-treated samples) independent biological replicates). Data were analyzed by a Tukey two-way ANOVA, using a mixed effect analysis (n.s. = not significant, * p = 0.0157, **** p < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Cells were incubated with primary antibodies against BRCA2 (R&D Systems, USA, Cat# MAB2476, RRID:AB_2259370, 1:100) and DPP9 (Ruth Geiss-Friedlander—University of Freiburg Cat# RGF_1, RRID:AB_2889071, 1:100) for 90 min at 37 °C and actin filaments were simultaneously counterstained with CytoPainter Phalloidin‐iFluor 488 Reagent (Abcam, United Kingdom).

Techniques: Residue, Blocking Assay, Labeling, Inhibition, Negative Control, Control

Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for western blot analysis: 53BP1 (Bethyl Laboratories, A300-272A, 1:3000), LIN37 (Santa Cruz Biotechnology, sc-515686, 1:200), BLM (Bethyl Laboratories, A300-572A, 1:2000), BRCA1 for mouse (R and D Systems, gift from Dr. Andre Nussenzweig, NCI, 1:1000) , BRCA1 for human (Millipore Sigma, 07-434, 1:1000), RAD51 (Millipore Sigma, ABE257, 1:2000), BARD1 (Thermo Fisher Scientific, PA5-85707, 1:1000), CtIP (gift from Dr. Richard Baer, [Columbia University, New York], 1:1000), MRE11 (Novus Biologicals, NB100-142, 1:2000), RIF1 (Abcam, ab13422, 1:500), SHLD1/C20orf196 (Thermo Fisher Scientific, PA5-559280, 1:200), GAPDH (Sigma, G8795, 1:10,000), KAP1 (Genetex, GTX102226, 1:2000), FANCD2 (R and D Systems, MAB93691, 1:1000), BRCA2 for human (Proteintech, 19791-1-AP, 1:500), Rb1 (Thermo Fisher Scientific, LF-MA0173, 1:1000), Phospho-Rb (Ser780) (Cell Signaling Technology, 8180T, 1:1000), Phospho-Rb (Ser807/811) (Cell Signaling Technology, 8516T, 1:1000), PCNA (Bethyl Laboratories, A300-276A, 1:3000), CDK4: (Novus Biologicals, NBP1-31308, 1:1000), CDK4 (phosphor Thr 172) (GeneTex, GTX00778, 1:1000), and RPA (Cell Signaling Technology, 2208S, 1:1000).

Techniques: Recombinant, Plasmid Preparation, Genome Wide, CRISPR, Clone Assay, Flow Cytometry, Staining, Sequencing, Software, Microscopy