Anti cleaved caspase 6

The whole mouse testis was homogenized in cold RIPA buffer (Cell Signaling Technology, #9806) containing 1 mM benzylsulfonyl fluoride (PMSF) (WAKO, #164-12181) and protease inhibitor cocktail (Complete EDTA-free, Roche, #11873580001). Lysates were centrifuged at 15,000 rpm for 15 min at 4 °C and supernatants were used for western blot analysis. Western blot analyses were performed with anti-phosphorylated AMPK (Cell Signaling technology, 1:1000; #2535), anti-αAMPK (Cell Signaling technology, 1:1000; #2532), anti-AdipoR1 (Immuno-Biological Laboratories, 1:1000; #18993), anti-cleaved caspase-3 (Cell Signaling technology, 1:1000; #9661), anti-cleaved caspase-6 (Cell Signaling technology, 1:1000; #9761), anti-cleaved caspase-9 (Cell Signaling technology, 1:1000; #9509), anti-α-tubulin (Cell Signaling technology, 1:4000; #2125) and HRP-linked anti-rabbit IgG (Cell Signaling technology, 1:2000–1:20000; #7074) antibodies. Uncropped western blot images are shown in Fig. S2-S5.

We purchased p,p’-DDT (1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane; product number 31041-100MG) from Sigma-Aldrich (www.sigmaaldrich.com), and propidium iodide from Abcam (www.abcam.cz: ab14085). For the western blot analysis, we used the following primary and secondary antibodies: anti-cleaved caspase-6 (#9761), anti-cleaved caspase-7 (#9491), anti-cleaved caspase-8 (#9496), anti-cleaved caspase-9 (#9505) anti-cleaved and total PARP (#9542), anti-GRP78 (#3177), and anti-CHOP (#2895) from Cell Signaling Technology (www.cellsignal.com). We purchased anti-actin (clone AC-40) primary antibody from Sigma-Aldrich (www.sigmaaldrich.com: A4700), and corresponding horseradish peroxidase-conjugated secondary antibodies from Proteintech (www.ptglab.com: SA00001-2, and SA00001-1).

TAS4464 was designed and synthetized at Taiho Pharmaceutical Co., Ltd. and cytarabine (cytosine beta-D-arabinofuranoside hydrochloride) and the anti-Actin antibody (#A2066) were purchased from Sigma-Aldrich Co., LLC. Anti-cleaved caspase-3 (#9664), anti-cleaved caspase-6 (#9761), anti-cleaved caspase-7 (#8438), anti-cleaved caspase-8 (#9496), anti-cleaved caspase-9 (#9501, #20750), anti-cleaved PARP (#9541), anti-Bak (#3814), anti-Bax (#2772), anti-Mcl-1 (#4572), anti-Puma (#4976), anti-Bim (#2819), anti-Bcl-2 (#2872), anti-Bcl-xl (#2762), anti-Bcl2l10 (#3869), anti-XIAP (#2042), and anti-c-Myc (#5605) antibodies were purchased from Cell Signaling Technology, Inc. Anti-c-FLIP (sc-5276), anti-FAS (sc-715), anti-FAS-L (sc-957) and anti-c-Myc (sc-40) antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-NEDD8 (ab81264), anti-NOXA (ab13654), anti-c-Myc (ab32072), and anti-α-Tubulin (ab4047) antibodies were purchased from Abcam plc.

Total mouse brain proteins were extracted using Western-IP Lysis Buffer (Beyotime,Wuhan, China) and quantified using the BCA protein assay kit (Beyotime, Wuhan, China). In total, 10 μg of total proteins were electrophoresed on 12% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked with PBS/Tween 20® (PBST) containing 5% non-fat milk for 90 min at room temperature and then incubated overnight at 4 °C with the following primary antibodies: anti-caspase-3, anti-cleaved caspase-3, anti-caspase-6, anti-cleaved caspase-6, anti-RIP3, anti-Beclin-1 (Cell Signaling Technology, Danvers, USA), anti-caspase-4, anti-phosphor-RIP3 (Abcam, Cambridge, UK), anti-LC3B (Beyotime,Wuhan, China), and anti-β-actin (Cell Signaling Technology, Danvers, USA) as the control. After incubation with HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-rat IgG (Cell Signaling Technology), the nitrocellulose membranes were washed three times, and bands were visualized by chemiluminescence with Pierce Enhanced Chemiluminescence Detection Reagent (Millipore, Burlington, USA) on the ChemiDoc™ Touch imaging system (Bio-Rad, Hercules, USA). Relative band intensity was analysed by AlphaViewTM software (Alpha Innotech Corporation, San Jose, USA).