Anti atf4

OPCs were washed twice with PBS and lysed in ice-cold RIPA buffer (Sigma) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, PI78441). Lysates were then centrifuged at 12,000 rpm for 20 min at 4°C. A total 20 µg of protein lysates was separated by 4–12% SDS-PAGE (Bio-Rad, 4561095) and transferred to a nitrocellulose membrane. The following primary antibodies were used: anti-p-eIF2α (Abcam, ab32157, 1:2000), anti-T-eIF2α (Cell Signaling, 9722s, 1:1000), anti-puromycin (Millipore, MABE343, 1:2000), anti-BIP (Cell Signaling, 3177s, 1:1000), anti-GADD34 (Proteintech, 10449–1-AP, 1:500), anti-ATF4 (Santa Cruz, sc-390063, 1:500), anti-CHOP (Thermo Fisher, MAI-250, 1:500), anti-XBP-1-spliced (Cell Signaling, 82914s, 1:1000), and anti-actin (Sigma, A2066, 1:2000). Quantification of Western blot bands were performed by Image Lab Software (Bio-Rad).

The natural product library was provided by Korea Institute of Science and Technology (KIST; Gangneung, Korea). The extract library was composed of ethanolic extracts of Korean native plants. MTT (tetrazolium bromide), dimethyl sulfoxide (DMSO), and N-Acetyl-L-Cysteine (NAC) were purchased from Sigma (St. Louis, MO). 2′,7′-Dichlorofluorescein diacetate (DCF-DA) was purchased from Molecular Probes (Eugene, OR). The TNF-α was purchased from Peprotech (Rocky Hill, NJ). The anti-PARP-1, anti-ATF4, anti-caspase-7, anti-actin, and anti-GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-CHOP, anti-phospho-NF-κB, anti-NF-κB, anti-IL-1β, anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho-eIF2α, anti-eIF2α, and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA). Annexin V-flamma 488 was purchased from Bioacts (Korea). Hoechst 33342 was purchased from Invitrogen (CA).

Total protein was extracted from ovarian tissues, mixed with sodium dodecyl sulfate (SDS) sample buffer, and then boiled for 10 mins. For protein analyses, 30 μg of protein from each sample was loaded onto an SDS polyacrylamide gel for electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes. The membranes were blocked in 5% skim milk for 60 mins at room temperature before incubation overnight at 4°C with the following primary antibodies: anti-ATF4 (1:1,000, Santa Cruz, USA, RRID: AB_2058752), anti-COX2 (1:1,000, Proteintech, China, RRID: AB_2085127), and anti-GAPDH (1:5,000, Abcam, USA, RRID: AB_2107448). The samples were then incubated for 90 mins at room temperature with secondary antibodies (1:5,000, Cell Signaling, USA). Chemiluminescence reagent (Thermo Fisher Scientific, USA) was used to visualize the blots. The representative blots were obtained from three independent experiments.

Western blotting was performed according to standard procedures. We used the following primary antibodies: anti-HOOK1 (Abcam, ab151756), anti-CASP3 (Cell Signaling, #9664), anti-CASP9 (Cell Signaling, #9502), anti-PARP (Cell Signaling, #9532), anti-p-H2AX (Ser139) (Cell Signaling, #9718), anti-NANOG (Santa Cruz Biotechnology, sc-293,121), anti-OCT3/4 (Santa Cruz Biotechnology, sc-5279), anti-KLF4 (Abcam, ab72543), anti-ATF6α (Santa Cruz Biotechnology, sc-166,659), anti-ATF4 (Santa Cruz Biotechnology, sc-390,063), anti-GRP78 (Santa Cruz Biotechnology, sc-13,539), anti-CHOP (Cell Signaling, #2895), anti-LC3B (Abcam, ab48394), anti-p62 (Abcam, ab109012), and anti-B-actin (Abcam, ab16039) as a loading control. We used the following secondary antibodies: rabbit anti-mouse (Abcam, ab97046) and goat anti-rabbit (Abcam, ab97051). The proteins were detected using an ECL detection system (Amersham Biosciences) and a Bio-Rad Chemidoc Touch.

The following antibodies and chemicals were purchased: anti- Cullin-4B, anti-FLAG (M2), and anti-α-tubulin antibodies, mouse IgG-agarose, monoclonal anti-HA-agarose, and anti-FLAG M2 affinity gel from Sigma-Aldrich Co. (St. Louis, MO); anti-caspase-3, anti-caspase-9, anti-cleaved caspase-3, and anti-cleaved caspase-7 antibodies from Cell Signaling Technology (Beverly, MA); anti-Apaf-1 antibody from Millipore (Temecula, CA) and Abcam (Cambridge, United Kingdom); anti-multi ubiquitin antibody (FK2) from Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan); anti-p62 (C-terminal) antibody (specific to amino acids 421–440 of human p62 protein) from PROGEN Biotechnik GmbH (Heidelberg, Germany); anti-HA (HA.11 and Y-11) and anti-myc antibodies from Covance (Richmond, CA) and Santa Cruz Biotechnology (Santa Cruz, CA); and anti-ATF4, anti-REST, and anti-Bcl-xS/L antibodies from Santa Cruz Biotechnology. HRP-conjugated anti-rat, anti-mouse, anti-rabbit, and anti-guinea pig IgG (H + L) antibodies were purchased from Southern Biotech (Birmingham, AL); MG132 from Calbiochem (La Jolla, CA); etoposide, E64d, pepstatin A, and cycloheximide from Sigma-Aldrich Co. All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan), Kanto Chemical (Tokyo, Japan), and Sigma-Aldrich Co.