Anti dna pkcs

FaDu ATM KO cells were analysed for pharmacodyanamic biomarker modulation by western blotting. Protein lysates were generated by adding radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% sodium dodecyl sulfate (SDS) and protease and phosphatase inhibitor tablets (Roche, Switzerland) to FaDu ATM KO cells 2 hours following the start of compound treatment in the second 24-hour treatment cycle. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher, USA). Proteins were separated by SDS-PAGE on NuPAGE 4% to 12% Bis-Tris (Thermo Fisher, USA) and transferred onto nitrocellulose membranes using the iBlot dry blotting system (Thermo Fisher, USA). Membranes were blocked with 5% milk in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T) and incubated with primary antibody overnight at 4°C. Primary antibodies used were anti-DNA-PKcs pS2056 (in-house generated), anti-DNA-PKcs (Cell Signalling, #12311, USA), and anti-vinculin (Sigma, 19191, USA). Membranes were then washed with TBS-T and incubated with HRP-conjugated anti-rabbit or anti-mouse antibodies for 1 hour at room temperature. Proteins were detected by incubating membranes with SuperSignal Dura extended-duration substrate (Thermo Fisher, USA) and visualised using the G:BOX ChemiGenius imaging system (Syngene, UK). Band intensities were quantified using Image J software.

MTT, dimethyl sulfoxide (DMSO), PI, DAPI and DCFH-DA were purchased from Sigma (St Louis, MO). RIPA buffer and JC-1 staining kit were purchased from Beyotime Biotechnology, China. The following antibodies were used: anti-PUMA, anti-p53, anti-Chk1, anti-Chk2, anti-p-Chk1(Ser345), anti-p-Chk2 (Thr68), anti-ATM, anti-p-ATM (Ser1981), anti-PARP, anti-GAPDH and anti-β-actin (Santa Cruz Technology, Santa Cruz, CA, USA); Anti-NOX1, anti-NOX4, anti-DUOX1 and anti-SOD1, anti-Keap1, anti-Nrf2, anti-HO-1, anti-DNA-PKcs, anti-p-DNA-PKcs (Thr 2609), anti-Caspase-3 and anti-Caspase-9 (Cell Signaling Technology, Danvers, MA, USA); Anti-γ H2AX (Ser139), anti-H2AX and anti-JNK, anti-p-JNK, anti-BCL-XL, anti-MCL-1 and anti-BCL-2 (Abcam). Rhodamine (TRITC) AffiniPure Goat anti-Rabbit IgG was from Santa Cruz Biotechnology; KU-60019, VE-821 and NU7026 were obtained from Selleck (USA). SP600125 were obtained from Calbiochem (La Jolla, CA, USA). Z-VAD-fmk was purchased from R&D Systems (Minneapolis, MN). These inhibitors were dissolved in DMSO and diluted to appropriate concentrations with cell culture media. Mitotracker and MitoSOX red mitochondrial superoxide indicator was purchased from Molecular Probe (USA).

Anti-NF-κB, anti-Bcl-2, anti-cyclin A, anti-cyclin B, anti-cyclin E, anti-phospho-Raf-1, anti-Akt, anti-Snail, and anti-β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-cleaved PARP, caspase-3, anti-ATM, anti-ATR, anti-Rad50, anti-p95/NBS1, anti-Rad52, anti-MRE11, anti-Ku70, anti-Ku80, anti-DNA-PKcs, anti-ERCC1, anti-Rad51, anti-Ras, anti-MEK1/2, anti-phospho-MEK1/2, anti-Erk1/2, anti-phospho-Erk1/2, anti-phospho-Akt, anti-vimentin, anti-E-cadherin, and anti-MMP9 were purchased from Cell Signaling Technology (Danvers, MA, USA), and anti-γ-H2AX was obtained from Millipore (Billerica, MA, USA). ZOL was purchased from Sigma-Aldrich (St. Louis, MO, USA). For in vitro experiments, ZOL was dissolved in PBS to make a 2 mmol/L stock solution and stored at −20°C.

Carfilzomib was obtained from Selleck Chemicals. Pan-histone deacetylase (HDAC) inhibitors panobinostat (PS) and vorinostat (VS) were obtained from Novartis Pharmaceuticals (East Hanover, NJ) and Selleck Chemicals, respectively. All drugs were prepared as 10 mM stocks in 100% DMSO and stored in small aliquots at −80°C to prevent multiple free thaw cycles. Anti-phosphorylated (p)-ATR (S428), anti-p-CHK1(S345), anti-α/β tubulin, anti-DNA-PKcs, anti-acetyl histone H3 (K9/K14), anti-histone H3, anti-RAD52, anti-BRCA2 and anti-acetyl lysine antibodies were purchased from Cell Signaling Technology (Berverly, MA). Anti-hsp90α and anti-hsp70 antibody was purchased from Enzo Biosciences (Plymouth Meeting, PA). Anti-BRCA1 and anti-γ-H2AX antibodies were obtained from Millipore (Billerica, MA). Anti-CHK1, anti-ATR, anti-HDAC1, anti-HDAC2, and anti-HDAC3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin, anti-FLAG, anti-acetylated α-tubulin and anti-GFP antibodies and short hairpin RNAs against HDAC1, HDAC2 and HDAC3 were purchased from Sigma-Aldrich (St. Louis, MO). Acetylated-K69 hsp90 (Ac-K69 hsp90) antibody was previously described [24 (link)]. Anti-c-RAF antibody was purchased from BD Transduction Labs (San Jose, CA).