Anti caspase 7

Whole cell lysates were prepared and analyzed by Western blotting as described previously [32 (link)]. Proteins in cell lysates were separated by precast 8-20% SDS-polyacrylamide gel electrophoresis, and then electrophoretically transferred from the gel onto polyvinylidene difluoride membranes. After blocking, blots were incubated with anti-GRP78, anti-GRP94, anti-phospho-PERK, anti-caspase-12, anti-caspase-7, anti-CHOP, anti-PARP, and anti-β-actin antibodies (Santa Cruz Biotechnology) and anti-eIF2α (Cell Signaling Technology, Danvers, MA, USA) in PBS within 0.1% Tween 20 for 1 h followed by two 15 min washes in PBS with 0.1% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 60 min. Detection was performed with Western blotting reagent ECL (Amersham-GE Healthcare Life Sciences, Pittsburgh, PA, USA), and chemiluminescence was exposed by the Kodak X-Omat films.

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).

Reagents were purchased from Sigma-Aldrich unless specified. Drugs were obtained from Sigma-Aldrich (etoposide, actinomycin D, NU6027), Enzo Life Sciences (zVAD-fmk), Tocris (KU55933, NU7441, SB218078), or Selleck (Hesperadin). Primary antibodies were anti–γ-H2AX (pS139; Santa Cruz; sc-101696); anti-Actin (Sigma; A2066); anti-TRF2 (clone 4A794) (Millipore; 05-521); anti-caspase-3 (BD Transduction Laboratories; cat. 610322); anti-caspase-7 (Santa Cruz; sc-8510); anti-CAD (Thermo; PA5-19913). Secondary antibodies were HRP-linked anti-mouse, anti-goat or anti-rabbit (Biorad); Alexa-488–conjugated anti-mouse or anti-rabbit (Invitrogen); Alexa-594–conjugated anti-mouse or anti-rabbit (Invitrogen).