Ab32566

Cells (1 × 10⁶) were lysed in RIPA lysis buffer (Solarbio Science & Technology Co., Ltd., Beijing, China) to collect total protein, and the protein concentration was examined using a bicinchoninic acid kit (Keygen Biotech Co., Ltd., Nanjing, Jiangsu, China) according to the instructions. An equal amount (60 μg) of protein sample was run on 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After being blocked in 5% not-fat milk for 1 h, the membranes were hybridized with the primary antibodies GAPDH (1: 10,000, ab181602, Abcam), GTSE1 (1:1000, GTX66223, GeneTex, CA, USA), Cyclin D1 (1:1000, #2978S, Cell Signaling Technology (CST), Beverly, MA, USA), Cyclin E1 (1:1000, #20808S, CST), proliferating cell nuclear antigen (PCNA; 1:1000, #13110S), cleaved caspase 3 (1:500, ab32042), Bax (1:1000, GTX109683, GeneTex), γH2AX (1:5000, ab81299), and DNA-PKcs (1:1000, ab32566) at 4 °C overnight, and then with HRP-conjugated secondary antibody (1:5000, ab205718, Abcam Inc., Cambridge, MA, USA) at room temperature for 1 h. The protein bands were developed using the ECL reagent (Pierce, Rockford, IL, USA).

Exponentially growing cells were harvested with trypsin, counted and 10⁶ cells were resuspended in 100 µL of PBS containing protease inhibitors. 100 µL of 2×Laemmli buffer (Bio-Rad) was added and samples were boiled at 95 °C for 10 min. Samples were separated with 4–20% gradient SDS–PAGE, transferred to the PVDF membrane, and blocked in 5% milk-TBST for 2 h at room temperature. Membranes were then incubated overnight at +4 °C with rabbit monoclonal antibodies anti-DNA PKcs (Abcam, ab32566, 1:1000), rabbit monoclonal antibodies anti-Sirt6 (CST, #12486, 1:1000) or rabbit polyclonal antibodies anti-Histone H3 (Abcam, ab1791, 1:10,000) in 5% BSA-TBST. After three washes for 10 min with TBST, membranes were incubated for 1 h at room temperature with goat anti-rabbit IgG H&L (HRP) (Abcam, ab6721, 1:5000). After three washes with TBST signal was developed with Clarity Western ECL substrate (Bio-Rad). The images were quantified with Image Lab (Bio-Rad). All blots shown were derived from the same experiment and were processed in parallel.

Antibodies used in this study are listed as follows: DNA-PKcs (ab32566, Abcam, UK, 1:5000), phosphorylated DNA-PKcs (ab124918, Abcam, 1:5000), phosphorylated ATM (ab81292, Abcam, 1:5000), ATM (ab32420, Abcam, 1:5000), PI3KCA (ab124918, Abcam, 1:1000), phosphorylated histone H2AX (2577, Cell Signaling Technology, USA, 1:500 for IB, 1:100 for IHC), H2AX (7631, Cell Signaling Technology, 1:1000), IRF9 (76684, Cell Signaling Technology, 1:1000), eIF-2α (5324, Cell Signaling Technology, 1:1000), phosphorylated eIF-2α (3398, Cell Signaling Technology, 1:1000), JNK (9252, Cell Signaling Technology, 1:1000), phosphorylated JNK (9255, Cell Signaling Technology, 1:1000), PERK(3179, Cell Signaling Technology, 1:1000), CHOP (2895, Cell Signaling Technology, 1:1000), Ki-67 (9449 s, Cell Signaling Technology, 1:400), cleaved-Caspase-3 (9664 s, Cell Signaling Technology, 1:1000 for IB, 1:500 for IHC), Caspase-3 (9662, Cell Signaling Technology, 1:1000), CD4 (25229, Cell Signaling Technology, 1:200), CD8 (98941, Cell Signaling Technology, 1:400), GAPDH (AP0063, Bioworld, USA, 1:10000), α-tubulin (ARG65693, Arigo Biolaboratories, China, 1:5000), β-tubulin (AP0064, Bioworld, 1:10000), M1 E1 and NS3 (produced by Beijing Protein Innovation, China, 1:2000). Anticancer compounds used in this study were purchased from Selleckchem.

Proteins were extracted from the suitably treated cells using RIPA lysis buffer (Solarbio, Beijing, China) and separated by 10% SDS-PAGE as previously described [16 (link)]. The blots were probed with primary antibodies targeting the following proteins: p-Sp1 (Ser101) (1:1000, Active Motif, Carlsbad, CA, USA), DNA-PKcs (1:1000, ab32566, Abcam, Cambridge, UK), DNA-PKcs (phospho S2056) (1:1000, ab124918, Abcam, Cambridge, UK), γH2AX (Ser139) (1:1000, Cell Signaling Technology, Boston, MA, USA), Sp1 (1:3000, Proteintech Group, Wuhan, China) and GAPDH (1:10,000, Proteintech Group, Wuhan, China). Protein expression was quantified using the Quantity One software (Version 4.6.2, Bio-Rad, Hercules, CA, USA).

Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) was performed using a Plus TUNEL Assay Kit (C10617, Invitrogen). Mitochondrial membrane potential was analyzed using a MITO-ID® membrane potential detection kit (ENZ-51018-K100, AmyJet Scientific Inc., Hubei, China) 64 (link). Western blots were performed as our previously described. 55 (link). DNA-PKc (ab32566, Abcam) and Fis1 (ab229969, Abcam) was used in our study.