Dna pkcs

The primary antibodies against the following proteins were used: GFP at 1:3000 (Abcam, ab290, ab69314), NIPBL I at 1:1000 (Enervald et al., 2013 (link)), NIPBL II at 1:500 (Santa Cruz, sc-374625) (Zuin et al., 2014 (link)), α-tubulin at 1:1000 (Sigma, T9026), γH2AX at 1:1000 (Millipore, 05-636), MAU2 at 1:200 (Visnes et al., 2014 (link)), HP1γ at 1:1000 (Millipore, 05-690 and Abcam, ab10480), RAD18 at 1:2500 (Abcam, AB57447), cyclin B1 at 1:400 (Santa Cruz Biotechnology, SC-245), RNF8 at 1:200 (Santa Cruz Biotechnology, SC-271462), RNF168 at 1:1000 (Millipore, ABE367), KAP1 at 1:1000 (Nordic Biosite, A300-274A), KAP1 phosphorylated at S824 at 1:1000 (Nordic Biosite, A300-767A), β-actin at 1:1000 (Abcam, ab8224), Chk1 at 1:1000 (Cell Signaling Technology, #2360), Chk1 phosphorylated at S345 at 1:1000 (Cell Signaling Technology, #2348), DNA-PKcs at 1:1000 (Thermo Fisher, MA5-13404), DNA-PKcs phosphorylated at S2056 at 1:1000 (Abcam, ab18192), poly(ADP-ribose) at 1:1000 (Enzo Life Sciences, 10H). All antibodies were previously validated by us or the respective manufacturer.

After the cells were treated with different concentrations of HS-173 and irradiated for various times, they were collected and washed with cold phosphate-buffered saline (PBS). The cells were then lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX, USA). Proteins in whole-cell lysates were resolved by sodium dodecyl sulfate protease and phosphatase inhibitor (SDS-PAGE), and transferred onto nitrocellulose membranes. The blots were first incubated with the appropriate primary antibodies, and then with horseradish peroxidase (HRP)-conjugated secondary antibodies. Antibody binding was detected using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA). Primary monoclonal antibodies against the following proteins were used: cleaved PARP, cleaved caspase-3, survivin, p-AKT, AKT (Cell Signaling Technology, MA, USA) p-Cdc2, p-ATM, ATM, p-DNA-PKcs, DNA-PKcs, p-KAP1, p-53BP1 (Abcam, Cambridge, UK) KAP1, 53BP1(Santa Cruz, Dallas, TX, USA) and β-actin (Sigma-Aldrich, OH, USA) Secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Cells were lysed in RIPA with 1 mM PMSF for 30 min on ice. The mixture was centrifuged at 14,000 g for 5 min and the precipitate was discarded. BCA protein assay (Beyotime, China) was performed to measure the protein concentration. Samples containing equal amount of protein were separated by SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membrane using standard procedure. Non-specific sites were blocked with 5% nonfat milk for 1 hour at room temperature. PVDF membrane was incubated at 4°C overnight with corresponding primary antibodies, Nanog(Cell Signaling Technology, Beverly, MA, USA), Oct3/4(Cell Signaling Technology, Beverly, MA, USA), STAT3(Cell Signaling Technology, Beverly, MA, USA), ABCG2(Cell Signaling Technology, Beverly, MA, USA), BCL-2(Cell Signaling Technology, Beverly, MA, USA, Beverly, MA, USA),and DNA-PKcs(Abcam, Cambridge, UK). After washed in TBS with 0.1% Tween 20, blots were incubated with anti-rabbit or anti-mouse secondary antibodies conjugated with horseradish peroxidase. Immunoreactive bands were detected by enhanced chemiluminescence(ECL)(GE Healthcare, UK).

After being washed with ice-cold PBS, cells were lysed with RIPA Lysis Buffer (VWR Life Science, Philadelphia, PA, USA), supplemented with Phosphatase Inhibitor Cocktail 2 (Sigma, St. Louis, MO, USA) and Protease Inhibitor Cocktail (Bimake, Houston, TX, USA). The Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) was used to determine protein concentration. Equal protein lysates were run on Novex 3–8% Tris-acetate 15 Well Mini Gels (Invitrogen, Carlsbad, CA, USA) and transferred to Immobilon-P membranes (Millipore, Burlington, MA, USA). Membranes were then probed with the following primary antibodies: GAPDH (Santa Cruz Biotechnologies, Dallas, TX, USA), DNA-PKcs (abcam, Cambridge, UK), phospho DNA-PKcs S2056 (abcam), Artemis (abcam), phospho Artemis S516 (abcam), XRCC4 (Santa Cruz Biotechnologies), Ligase IV (abcam), CD4 (abcam), and DYKDDDDK (FLAG) Tag (Invitrogen). After exposure to the matching HRP-conjugated secondary antibody, cells were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).