Caspase 6

Total proteins extracted from cells or tumor tissues were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins in the gel were electrotransferred onto polyvinylidene fluoride membranes. The proteins were then probed with antibodies against DR6 (BioVision, Milpitas, CA, USA), IL-6R (Proteintech, Rosemont, IL, USA), VEGF-R-2 (Abcam, Cambridge, MA, USA), VEGF-D and PDGFR-α (Biogot, Nanjing, China), caspase 3, caspase 6, p-IκBα, p-P38, P38, p-STAT3 or STAT3 (Cell Signaling Technology, Danvers, MA, USA) and then incubated with an appropriate horse radish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology). The target proteins were visualized with a chemiluminescence system (Millipore, MA, USA) and exposed to X-ray film. The blot images were scanned and processed using Microsoft Office Picture Manager. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (KangChen Biotech, Beijing, China) was used as an internal control.

For Western blotting, total protein extracts were obtained by cell lysis buffer containing 150 mM NaCl, EDTA (1 mM), 1% NP-40; 50 mM Tris (pH 8.0), as well as phosphatase and protease inhibitors. Following SDS polyacrylamide gel electrophoresis, proteins were blotted on nitrocellulose membranes.
Primary antibodies of Cell Signaling (Danvers, MA, USA): Caspase-3 (9662, rabbit, 1:1000), Cleaved Caspase-3 (9664, rabbit, 1:1000), Caspase-8 (9746, mouse, 1:1000), Caspase-9 (9502, rabbit, 1:1000), Caspase-6 (9762, rabbit, 1:1000), Caspase-7 (9492, rabbit, 1:1000), XIAP (2042, rabbit, 1:1000), Mcl-1 (4572, rabbit, 1:1000), Bad (9292, rabbit, 1:1000), and Bcl-w (2724, rabbit, 1:1000). Primary antibodies of Santa Cruz Biotech (Dallas, TX, USA): c-FILP (sc-5276, mouse, 1:500), survivin (sc-177779, mouse, 1:500), p21(sc-6246, mouse, 1:500), p53 (sc-126, mouse, 1:500), β-actin (sc-47778, mouse, 1:1000), Puma (sc-374223, mouse, 1:500), Bax (sc-7480, mouse, 1:500), and Bak (sc-832, mouse, 1:500). Primary antibody of Abcam (Cambridge, UK): DR5 (ab8416, rabbit, 1:1000). Secondary antibodies: peroxidase-labelled goat anti-rabbit and goat anti-mouse (Dako, Hamburg, Germany; 1:5000).

Samples were separated on 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA). For cytoplasmic fractions or whole cell lysates, 100 μg of total protein was loaded. For nuclear fractions, 10 μg of total protein was loaded. The following antibodies and conditions were used: cleaved PARP (9544; 1:500; Cell Signaling, Danvers, MA), caspase-6 (9762; 1:500; Cell Signaling), cleaved caspase-3 (9664; 1:500; Cell Signaling), XBP-1 (sc-7160; 1:200; Santa Cruz, Dallas, TX), ATF4 (sc-200; 1:200; Santa Cruz), ATF6 (IMG-273; 1:200; Imgenex, San Diego, CA); LRH-1 (PP-H2325-10; 1:200; Perseus Proteomics, Tokyo, Japan), pATF2 (9225S; 1:500; Cell Signaling [note: we have experienced difficulties with recent batches]), ATF2 (ab47476; 1:1000; Abcam, Cambridge, England); and PLK3 (4896S; 1:400; Cell Signaling). Immobilon Western substrate (Millipore, Billerica, MA) was used for detection.

WB analysis was performed as described in detail previously.^{28 (link), 61 (link)} For tissue analysis, rats were killed at 24 h after SAH induction, and the cortical samples were homogenized in RIPA buffer (Sangon, Shanghai, China) and centrifuged at 12 000 × g for 15 min at 4 °C. The supernatants were collected and protein concentrations were determined with a BCA Kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts (60 μg) of protein per sample were subjected to WB. The antibodies used include: GCN5 (CST, no. 3305), H2B (CST, no. 2722), H3 (CST, no. 9715), H4 (CST, no. 2592), Ac-H2BK5 (CST, no. 2574), Ac-H3K9 (CST, no. 9671), Ac-H3K14 (CST, no. 7627), Ac-H3K27 (CST, no. 4353), Ac-H4K12 (CST, no. 2591), Ac-H4K5 (CST, no. 8647), Caspase 3 (CST, no. 9662), Caspase 6 (CST, no. 9762), Bim (CST, no. 3305), E2F1 (CST, no. 3742), Egr-1 (CST, no. 4154), GAPDH (no. 2118) (Cell Signaling Technology; diluted at 1:1000), Flag, or tubulin (Sigma; both diluted at 1:10 000). The horseradish peroxidase-conjugated secondary antibodies are used (Jackson ImmunoRes, West Grove, PA, USA), and signals are visualized by using the ECL chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA).

LCTP-induced apoptosis in U87Mg cells was assessed by Western blot analysis of caspases 6 and Poly (ADP-ribose) polimesare (PARP). The samples were prepared as previously reported for cell cycle proteins analysis and the PVDF membranes were incubated with caspase 6 (Cell Signaling, 1:1000), and PARP (Cell Signaling 1.100) in 2.5% milk in TBST overnight at 4 °C. The incubation with secondary antibodies and detection of proteins were performed as above described.

Rotenone, DMC, thiobarbituric acid (TBA), MTT, 2–7-diacetyldichlorofluorescein (DCFH-DA), rhodamine 123 (Rh-123), heat-inactivated fetal calf serum (FCS), Dulbecco’s modified Eagle’s medium (DMEM), glutamine, penicillin–streptomycin, EDTA, and trypsin were acquired from Sigma-Aldrich (Bangalore, Karnataka, India. Anti- Bax, Bad, Bcl-xL, Bcl-2, caspase-3, caspase-6, and caspase-8, caspase-9, cyt-c (cytosol and mitochondria) anti-bodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA) and β-actin antibody were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). Anti-mouse and anti-rabbit secondary anti-bodies were purchased from Bangalore Genei Pvt. Ltd., (Bangalore, Karnataka, India). All other chemicals and biochemical used were of analytical grade.