Histone h1

Western blot analysis was performed as previously described 30 (link). Antibodies against PARP1 (R&D, McKinley Place NE Minneapolis, USA), PAR (Trevigen, Helgerman CT, Gaithersburg. USA), histone H1 (Santa Cruz Biotechnology, Santa Cruz, California, USA), β-actin (Santa Cruz Biotechnology, Santa Cruz, California, USA), GAPDH (Santa Cruz Biotechnology, Santa Cruz, California, USA) or LXRα (Abcam, Cambridge, England) were used as primary antibodies. Specific bands detected using a chemiluminescence assay (ECL detection reagents, Pierce, USA) were recorded onto X-ray film. BioRad Quantity One software (version 4.4) was used for quantification.
Far-western blot assays and AP-PARP1 protein were performed as described previously 17 (link). Membranes were incubated with 1µg/ml recombinant PARP1 protein (Trevigen, Helgerman CT, Gaithersburg. USA) or 1 µg/ml AP-PARP1 protein, 1 µg/ml recombinant protein LXRα (Abnova, Taiwan) or 1 µg/ml recombinant β-actin (Abnova, Taiwan).

Antibodies against HSF1, HSP70, HSP27, DNA-PKcs, Ku86, Ku70, β-actin, GST, HA, Histone H1, and p53 were purchased from Santa Cruz Biotechnology (Santa Cruz, diluted 1:1000). Antibodies against p-p53, p-DNA-PKcs (Ser 2056), γ-H2AX, FLAG, p-HSF1 (Ser 326), and Ku70 (N3H10) were obtained from Cell Signaling Technology, Abcam, Millipore, Sigma-Aldrich, and Thermo Scientific, respectively (diluted 1:1000). Pre-designed siRNAs for human HSF1, HSP27, HSP70, Ku70, Ku86, and a negative control Si-RNA (30 nM) were purchase from Bioneer Corporation. Propidium iodide (PI) and RNase A were purchased from Sigma-Aldrich.

HCT116 and SW480 colon cancer cells were treated with MMPP (0-15 μg/mL) for 24 h, then were homogenized with a protein extraction solution (PRO-PREP™, iNtRON Biotechnology, Seongnam, Gyeonggi, Korea), and lysed by 60 min incubation on ice. Western blotting was described elsewhere [36 (link)]. After a short washing in TBST, the membranes were immunoblotted with the following primary antibodies: caspase-3 and caspase-8 (1:1000 dilutions; Cell Signaling, Beverly, MA, USA) and Fas, DR3, DR4, DR5, DR6, IKKβ, p-IKKβ, IκBα, p-IκBα, p50, p65, Bcl-2, Bax, Histone-H1 and β-actin (1:1000 dilutions; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were performed using specific antibodies followed by second antibodies and visualization by chemiluminescence (ECL) detection system.

To prepare liver tissue samples for Western blotting, equal amounts of protein (30 μg) were extracted and resolved on 7–12% SDS-PAGE gels. The separated proteins were then transferred to PVDF membranes (Millipore) and incubated overnight at 4 °C with primary antibodies specific to the target proteins. The following day, the membranes were incubated with anti-mouse, anti-goat, or anti-rabbit secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. Primary antibodies used were COX-2 (sc-376861), iNOS (sc-7271), NF-κB p65 (sc-8008), p-ERK (sc-7383), p-p38 MAPK (sc-7973), p38 MAPK (sc-7972), p-Akt (sc-7985-r), Akt (sc-8312), PI3-kinase p110β (sc-602), Nrf2 (sc-722), HO-1 (sc-136961), AMPK (sc-25792), Histone H1 (sc-393358), and β-actin (Sc-47778), all purchased from Santa Cruz Biotechnology. Other primary antibodies used included ERK (#9102), p-JNK (#9255), JNK (#9252), and p-AMPK (#2535) from Cell Signaling Technology, and GAPDH (GTX100118) from Gene Tex. The blots were developed using an ECL detection kit (Advansta, CA, USA), and a quantitative analysis of protein levels was performed using ImageJ 1.53e software (NIH, Bethesda, MD, USA).

The human neuroblastoma cell line SK-N-SH as well as SK-N-SH-MJD26 and SK-N-SH-MJD78 cells stably expressing full-length ataxin-3 with 26 and 78 CAG repeats, respectively, were given by Prof. Mingli Hsieh (Department of Life Science, Tunghai University, Taiwan). The UAS-MJDtr-Q27, UAS-MJDtr-Q78, and elav-Gal4 fly strains were obtained from the Bloomington Drosophila stock center (Indiana University, Bloomington, IN, USA). CA, Res, and tBH were from Sigma Chemical Company (St Louis, MO). The specific antibodies for hsp27, p53, Bax, Bcl-2, beclin-1, histone H1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cytochrome c, and total and phosphorylated IKK-α/β and IκB-α were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against total (pro) and cleaved PARP, caspase-9, caspase-7, and phosphorylated p53 were from Cell Signaling Technology Inc. (Beverly, MA). The specific antibodies for p65, caspase-3, LC3 and β-actin were from BD Biosciences (Boston, MA), Novus Biologicals (Littleton, CO), and MBL International (Wobum, MA), EMD Millipore (Billerica, MA), respectively. Antibodies against p62 and ataxin-3 were obtained from Abcam (Cambridge, MA). The Mitochondria/Cytosol Fractionation Kit and Caspase-3 Colorimetric Assay Kit were from BioVision Inc. (San Francisco, CA, USA) and Millipore (Temecula, CA), respectively.

The brain tissues, treated astrocytes, and microglial BV-2 cells were prepared as previously described (Gu et al. 2015 (link)). An equal amount of total protein (20 µg) was resolved on 8–15% sodium dodecyl sulfate polyacrylamide gel and then transferred to a PVDF membrane (Immobilon-P; pore size 0.45 µm, EMD Millipore, USA). The membranes were blocked for 1 h in 5% skim milk solution and incubated for overnight at 4 °C with specific antibodies. To detect target proteins, specific antibodies against APP, iNOS (1:1000, Novus Biologicals, Inc., Littleton), BACE1, IBA-1 (1:1000, Abcam, Inc., Cambridge, MA, USA), COX-2 (1:1000, Cell Signaling Technology, Inc., Beverly, MA, USA), GFAP, p50, p65, IκB, phospho-IκB, β-actin, Histone H1 (1:1000, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) were used. The blots were then incubated with one of the following corresponding conjugated secondary antibodies: goat anti-rabbit, goat anti-mouse, or donkey anti-goat IgG-HRP (1:5000; Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA). Immunoreactive proteins were detected with an enhanced chemiluminescence Western blotting detection system. The relative density of the protein bands was scanned by densitometry using MyImage (SLB, Seoul, Korea) and quantified by Labworks 4.0 software (UVP Inc., Upland, CA, USA).