anti lig3  (R&D Systems)

Journal: Nature Aging

Article Title: Comparative analysis of senolytic drugs reveals mitochondrial determinants of efficacy and resistance

doi: 10.1038/s43587-025-01057-z

Figure Lengend Snippet: a , DXR-induced senescent TIG-3 cells were treated with ARV825 (25 nM) for 3 days and analysed by western blotting with the indicated antibodies. b, c , Early-passage (control) or DXR-induced senescent (DXR-Sen) TIG-3 cells were transfected twice at 3-day intervals with validated siRNAs against BRD4, or a control siRNA. Cells were subjected to western blotting ( b ) or MT-1 staining for mitochondrial membrane potential ( c ), β-actin was used as a loading control ( b ). Nuclei were counterstained with DAPI and histograms show single-cell quantification; 60 cells were analysed per group ( c ). d , DXR-induced senescent TIG-3 cells were analysed by western blotting after immunoprecipitation with the antibodies indicated at the top (IP). e , DXR-induced senescent TIG-3 cells were subjected to immunofluorescence staining for XRCC4 and MitoTracker; nuclei were counterstained with DAPI. f-k , Control and DXR-induced senescent TIG-3 cells were transfected twice at 3-day intervals with siRNAs against XRCC4, LIG3, or control siRNA. Cells were analysed by western blotting ( f, j ), cell survival assays ( g, k ), MT-1 staining for mitochondrial membrane potential ( h) , or CellROX staining for ROS levels ( i ). Nuclei were counterstained with DAPI. For h and i , histograms show single-cell quantification; 96 cells (control) and 51 cells (DXR-Sen) were analysed per group in h , and 58 cells per group in i . β-actin was used as a loading control ( f, j ). Data are presented as mean ± s.d. Statistical significance was assessed by two-sided Student’s or Welch’s t-test ( c , g , h , i , k ). Scale bars, 5 µm ( b , c , d , h ). Although data shown are from technical replicates( c, g, h, i, k ), experiments were independently repeated at least once to confirm reproducibility.

Article Snippet: After blocking with 5% milk, membranes were incubated with primary antibodies: β-actin (1:2,000 dilution; A5316, Sigma-Aldrich), lamin B1 (1:1,000 dilution; ab16048, Abcam), p16 (1:1,000 dilution; sc-56330, Santa Cruz), p21 (1:1,000 dilution; 2947, Cell Signaling Technology), XRCC4 (1:1,000 dilution; sc-271087, Santa Cruz), LIG3 (1:1,000 dilution; sc-135883, Santa Cruz), POLG (1:1,000 dilution; ab128899, Abcam), ATP6V0E1 (1:1,000 dilution; PA5-1114887, Thermo Fisher Scientific) and BRD4 (1:1,000 dilution; 13440, Cell Signaling Technology).

Techniques: Western Blot, Control, Transfection, Staining, Membrane, Single Cell, Immunoprecipitation, Immunofluorescence

Journal: bioRxiv

Article Title: Microhomology-mediated end joining acts directly on replication forks to repair single-ended double strand breaks

doi: 10.64898/2026.01.15.699632

Figure Lengend Snippet: MMEJ induced by Cas9 and Cas9n exhibits distinct genetic dependence. ( A-C ) MMEJ was assayed in U2OS (EGFP-MMEJ) cells expressing shRNAs for Polθ ( A ), LIG3 ( A ), RPA2 ( B ), MRE11 ( C ) or CtIP ( C ) after g2/Cas9 WT or g2/Cas9 D10A cleavage. ( D ) MMEJ was assayed in WT or POLQ -KO mES (EGFP-MMEJ) cells four days after transfection of plasmids encoding g2/Cas9 WT or g2/Cas9 D10A . ( E ) mES (EGFP-MMEJ) cells with POLQ WT allele or knock-in (KI) alleles containing mutations of K120G or D2494P/E2495R were assayed for MMEJ after g2/Cas9 WT or g2/Cas9 D10A cleavage. ( F ) In vitro biochemical assay showing Polθ-HelD activity in unwinding dsDNA to facilitate strand exchange with ssDNA carrying 15 bp homology. 32 P-5’-labeled ssDNA was mixed with the indicated dsDNA substrate and incubated with Polθ-HelD, ATP and/or RPA, and the reaction products were resolved on a non-denaturing gel. ( G ) In vitro biochemical assay showing strand displacement DNA synthesis by Polθ-HelD and Polθ-PolD. 32 P-5’-labeled ssDNA was mixed with the indicated dsDNA substrates and incubated with Polθ-HelD, ATP and/or RPA, followed by DNA extension with Polθ-PolD with 0.1 mM dNTPs, and the reaction products were resolved on a denaturing gel.

Article Snippet: Antibodies used in immunoblotting are: MRE11 (Cell Signaling Technology, 4895), CtIP (Proteintech, 12624-1-AP), RPA1 (Sigma-Aldrich, NA13), RPA2 (Bethyl, A300-244A), PCNA (Cell Signaling Technology, 13110), MCM2 (Proteintech, 10513-1-AP), DNA2 (Proteintech, 18727-1-AP), KU70 (Santa Cruz Biotechnology, sc-17789), HA (Santa Cruz Biotechnology, sc-7392), Flag (Sigma-Aldrich, F1804), BLM (Bethyl, A300-110A), EXO1 (Bethyl, A302-640A), ATR (Santa Cruz Biotechnology, sc-515173), BRCA1 (Santa Cruz Biotechnology, sc-6954), RAD51 (Santa Cruz Biotechnology, sc-398587), LIG3 (Proteintech, 26583-1-AP), γH2AX (Upstate, 07-164), Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs, 115-035-146), and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Labs, 111-035-144).

Techniques: Expressing, Transfection, Knock-In, In Vitro, Activity Assay, Labeling, Incubation, DNA Synthesis