Anti noxa

Immunoblotting was carried out as previously described [55 ]. The following antibodies were used in this study: anti-PARP-1, anti-cleaved caspase-3, anti-cleaved caspase-8, anti-caspase-9, anti-cleaved caspase-9, anti-HO-1, anti-NOXA, anti-CHOP, anti-GRP78, anti-PUMA (Cell signaling Technology, Beverly, MA), anti-actin (MP Biomedicals, Solon, OH), goat anti-rabbit IgG-horseradish peroxidase (HRP), and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Korean Red Ginseng powder was kindly provided by the Korea Ginseng Corporation (Daejeon, Korea). The KRG powders were dissolved in distilled water. N-acetyl-l-cysteine (NAC) and Anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-caspase-3, anti-caspase-9, anti-cleaved PARP, anti-BIM, anti-Noxa, anti-survivin, anti-IRE1α, anti-phospho IRE1α, anti-GRP94, anti-eIF2α, anti-phospho eIF2α, anti-ATF6, anti-PERK, anti-phospho PERK, anti-Bip anti-XBP1s, anti-ATF4, anti-SOD2, anti-SOD1, anti-catalase, anti-NOX4, and anti-NOX2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Bak, anti-BAX, anti-Bcl-2, anti-SOD3, and anti-CHOP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-mouse and Rabbit IgG HRP, the secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA).

Following treatment, the fibroblasts were harvested and lysed in RIPA buffer (Beyotime, Hangzhou, China) on ice. After the lysates were centrifuged in 4°C at 13,000 ×  g for 10 min, the supernatants were collected for Western blot analysis. The protein concentration was determined by the BCA Protein Assay Kit (Beyotime, Hangzhou, China). The proteins were separated by 6%–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). Following blocking with 5% skimmed milk in TBST for 2 h, the membranes were incubated with the appropriate primary and secondary antibodies successively according to the instructions. The primary antibodies used were anti-NOXA, anti-cleaved-caspase3, anti-cleaved-poly ADP-ribose polymerase (cleaved PARP), anti-caspase3, anti-PARP, anti-Bax, anti-Bcl-2 and anti-β-actin antibodies (Cell Signaling Technology, Beverly, MA, USA). The anti-mouse or anti-rabbit IgG were also purchased from Cell Signaling Technology.

Cells were lysed in 1× SDS sample buffer supplemented with the protease inhibitor mixture (Sigma‐Aldrich, St Louis, MO, USA). Equal amounts of protein (30 μg) were separated on SDS/polyacrylamide gels and then transferred onto membrane filters (Merck Millipore, Amsterdam, the Netherlands). After blocking with 5% non‐fat dry milk, the membranes were probed with anti‐p53 (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐phospho‐p53 at Ser‐15 (Cell Signaling Technology, Danvers, CA, USA), anti‐acetyl‐p53 at Lys‐373/382 (Upstate, Lake Placid, NY, USA), anti‐p21^WAF1 (Santa Cruz Biotechnology), anti‐Bcl‐2‐associated X protein (BAX; Cell Signaling Technology), anti‐NOXA (Cell Signaling Technology), anti‐HDAC2 (Cell Signaling Technology), anti‐poly (ADP‐ribose) polymerase (PARP; Cell Signaling Technologies), anti‐γH2AX (BioLegend, San Diego, CA, USA), anti‐ATM (Santa Cruz Biotechnology), anti‐phospho‐ATM at Ser‐1981 (Merck Millipore) or with anti‐actin antibody (Santa Cruz Biotechnology) followed by an incubation with horseradish peroxidase‐conjugated secondary antibodies (Invitrogen). Immunodetection was performed with enhanced chemiluminescence (ECL; GE Healthcare Life Science, Piscataway, NJ, USA).

Fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Gibco/BRL Life Technologies (Grand Island, NY, USA). The anti-Bak, anti-Bcl-2, anti-PARP, anti-p53, anti-phospho-p53, anti-acetyl-p53, anti-Noxa, anti-PUMA, anti-TRAIL, anti-DR5, anti-C-Flip and anti-SIRT1 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-p21 and anti-Myc antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The anti-acetyl-Myc and β-actin antibody were from EMD Millipore, Inc. (Burlington, MA, USA). The antisera to tNOX used in our western blot analyses were generated as described previously [18 (link)]. The anti-mouse and anti-rabbit IgG antibodies and other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise specified.

Genipin was purchased from Calbiochem (San Diego, CA, USA). GANT61 was purchased from Selleckchem (Houston, TX, USA). Protein G PLUS-Agarose and anti-BAX, anti-BCL2, anti-MCL-1, anti-UB, anti-BCL-Xl, anti-GLI3, anti-PCAF, anti-SHH, anti-PTCH, and anti-p53 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-NOXA, anti-PUMA, anti-BIM, anti-SURVIVIN, anti-BID, anti-cleaved PARP, anti-CASP3, anti-CASP9, anti-β-TrCP, and anti-GLI1 antibodies were purchased from Cell Signaling (Beverly, MA, USA). The anti-actin antibody was obtained from Sigma (St. Louis, MO, USA), the anti-SMO antibody was obtained from Abcam (Cambridge, UK), and the anti-ITCH antibody was purchased from BD science (San Jose, CA, USA). The secondary antibodies, anti-mouse IgG HRP and anti-rabbit IgG HRP, were obtained from Cell Signaling.

MG132 was purchased from Selleckchem. Cycloheximide was purchased from Calbiochem. Phos-tag acrylamide was purchased from Wako. The TIAamp Virus RNA kit obtained from Tiangen was used to extract RNA from plasma. The following primary antibodies were used: anti-SDH5 (Cell Signaling Technology, USA), anti-P53 (Santa Cruz Biotechnology, USA), anti-MDM2 (Abcam, UK), anti-P21 (Cell Signaling Technology, USA), anti-γH2AX (Cell Signaling Technology, USA), anti-H2AX (Cell Signaling Technology, USA), anti-Noxa (Cell Signaling Technology, USA), anti-BCL2 (Cell Signaling Technology, USA), anti-BAX (Cell Signaling Technology, USA), anti-PUMA (Cell Signaling Technology, USA), anti-Caspase9 (Cell Signaling Technology, USA), anti-Cleaved Caspase 9 (Cell Signaling Technology, USA), anti-Caspase3 (Cell Signaling Technology, USA), anti-Cleaved Caspase3 (Cell Signaling Technology, USA), anti-DNA-PKcsThr2609 (ABclonal Biotech Co., China), anti-DNA-PKcs (ABclonal Biotech Co., China), anti-ku70 (ABclonal Biotech Co., China), anti-ku86 (ABclonal Biotech Co., China), anti-HA (Cell Signaling Technology, USA), anti-flag (Santa Cruz Biotechnology, USA), anti-GAPDH (Abcam, UK),anti-Actin (Cell Signaling Technology, USA), and anti-laminA (Santa Cruz Biotechnology, USA).