Anti dna ligase 4

Oligonucleotides used in this study are as follows; only one strand of the oligonucleotides used for targeting PAM sites are presented and are without BbsI overhangs.
For 293T Lig4 CRISPR experiments:
gRNA Lig4: CATACGTTCACCATCTAGCT
Lig4 K273A HDR:
CTATTGCAGATATTGAGCACATTGAGAAGGATATGAAACATCAGAGTTTCTACATAGAAACAGCGCTAGATGGTGAACGTATGCAAATGCACAAAGATGGAGATGTATATAAATACTTCTCTCGAAATGG
For U2OS CRISPR experiments:
gRNA Lig4: GTTCAGCACTTGAGCAAAAG.
The U20S clone used in the experiments had + 1 frameshift mutations on both alleles.
For 293T FANCG CRISPR experiments:
gRNA1 FancG:GGGCCAGGCCTGGGTTCAAC
gRNA2 FancG:GACTTAAGAGAAAGGGACTG
5′ FancG PCR: CCCAAGATGTCCCGGCTGTGGG
3′ FancG PCR:CCATGGGCCTCTCTGTCCTTGCAC
Oligonucleotides for substrate PCR:
5′ coding joint PCR: CGGTGGGAGGTCTATATAAGCA
3′ coding joint PCR:CTACACCGTGGTGGAGCAGTA
5′ signal joint PCR:ACCTTGAAGCGCATGAAGGGC
3′ signal joint PCR:TCCATGCGGTACTTCATGGTC
Antibodies utilized in this study include: anti-DNA Ligase IV (Abcam, 193353), anti-Ku80 (Invitrogen, 111), anti-DNA-PKcs (generous gift Tim Carter).

For SDS-PAGE/Western blot analysis, the protein lysates were prepared by lysis with Tris-HCl buffer, NP-40 (1 %), PMSF (1 mM) and inhibitor cocktail (Roche, Switzerland). Protein samples were separated by 4-20% SDS-PAGE (BioRad, Berkeley, California, USA) and transferred onto PVDF membrane. Protein detection was performed with polyclonal antibodies: anti-thymidylate synthase (Abcam, 1:200), anti-ATM (Abcam, 1:2000), anti-phospho (S1981) ATM (Abcam, 1:1000), anti-DNA Ligase IV (Abcam, 1:500), anti-PARP (Cell Signaling, 1:1000), anti-cleaved PARP (Cell Signaling, 1:1000), anti-Caspase-3 (Cell Signaling, 1:1000), anti-cleaved Caspase-3 (Cell Signaling, 1:1000). The secondary anti-rabbit IgG coupled to HRP (Cell Signaling, 1:1000) was visualized with enhanced chemiluminescence (ECL+, GE Healthcare, UK). Equal protein loading was controlled using GAPDH specific antibody (Ambion, Life Technologies, Carlsbad, California, USA) and secondary goat anti-mouse IgG linked to HRP (Abcam). The experiments were carried out three times in triplicates.

Cells were washed with PBS and lysed in ice-cold lysis buffer (140 mmol/l NaCl, 50 mmol/l EDTA, 10% glycerol, 1% Nonidet P-40, 20 mmol/l Tris HCl pH 7) containing protease inhibitors (Complete, Roche Applied Science, Indianapolis). Protein concentration was measured using the Bradford assay (BioRad, Hercules, CA). Protein samples (20 μg/lane) were subjected to SDS-PAGE and transferred to PVDF membrane (BioRad). After blocking, membranes were incubated with anti-human antibodies. The following primary antibodies were used: anti-Ku70 (A-9, mouse, Santa Cruz Biotechnology), anti-Ku86 (S10B1, mouse, Santa Cruz Biotechnology), anti DNA-PKcs (rabbit, Abcam, Cambridge, UK), anti-DNA ligase IV (rabbit, Abcam), Anti-Artemis (goat, Abcam), anti-XRCC4 (A-7, mouse, Santa Cruz Biotechnology), anti-DNA ligase IIIα (1F1, mouse, Gene Tex, Irvine, CA, USA), anti-WRN (H-300, rabbit, Santa Cruz Biotechnology), anti-Rad51 (Rabbit, Santa Cruz Biotechnology). Horseradish peroxidase–linked donkey anti-rabbit, anti-mouse or anti-goat antibodies (Santa Cruz Biotechnology) were used as secondary antibodies at 1:5,000 dilution. Immunoblots were incubated for 1h at RT and developed using enhanced chemiluminescence western blotting detection reagents (Amersham Biosciences, Piscataway, NJ).