Akta purifier

HONE-1 cells were treated with 50 μg/ml cycloheximide at 37°C for 30 min before harvest. Total cell lysates were clarified by centrifugation at 4°C for 10 min at 13,000 rpm. The supernatants were loaded on the top of a 10-50% sucrose gradient and ultracentrifuged in a SW41Ti Beckman rotor at 4°C for 4 h at 36,000 rpm. The fractions were collected from the bottom of the tubes, and the absorbance was measured at a wavelength of 254 nm using an AKTA purifier (GE Healthcare, Amersham Biosciences). RNA and protein were extracted using TRIzol reagent (Invitrogen). Protein expression and mRNA expression were further analyzed by western immunoblot analysis and semi-RT-qPCR, respectively.

Size exclusion chromatography (AKTApurifier, GE Healthcare Life Sciences, USA) was performed so as to achieve a highly pure protein38 . The chromatographic procedure was carried out using XK26-100 column packed with superdex 200 which had been equilibrated with phosphate buffer, pH 7.0. The employed flow rate was 2.2 mL/min. The results were analyzed by UNICORN software. And the fractions of gel filtration were investigated for the desired protein using SDS-PAGE procedure.

IIA mixed histones were diluted in HEPES-buffered saline (HBS, 10 mM HEPES pH 7.4, containing 150 mM NaCl) and applied to a HiTrap heparin-Sepharose column using an AKTA purifier (both GE Healthcare, Hatfield, UK). Histones were eluted over a continuous salt gradient (0–2 M NaCl) and fractions analysed by SDS-PAGE and Coomassie staining. This provided a means to separate the core complex from the H1 histone. Appropriate fractions were pooled and dialysed into HBS. Protein concentrations were determined with the Pierce BCA protein assay kit (ThermoFisher). Histone citrullination was performed by incubating histones (600 μg/ml) with recombinant PAD4 (25 nM) in citrullination buffer (10 mM HEPES pH 7.4, 4 mM CaCl₂, 4 mM DTT) for 16 h at 37 °C. Citrullinated histones were purified using HiTrap heparin-Sepharose chromatography as above, except that elution was performed in a single step with 2 M NaCl. Citrullination was confirmed by western blotting with anti-citrullinated histone H3 antibodies (ab5103, Abcam, Cambridge, UK). For digestion reactions, histones (600 μg/ml) were incubated with 250 nM activated protein C (APC, Xigris, Eli Lilly Indiana IN, USA), or 10 nM neutrophil elastase (Sigma Poole, Dorset, UK) for 16 h at 37 °C, and purified using HiTrap heparin-Sepharose, as described above.

The full-length (FL, 1.1 Kb) tau (CG31057) region was amplified from a pUAST-dtau vector (kind gift from Efthimios Skoulakis, Vari, Greece35 (link)) using the following primers: FL forward, 5′-CACCATGGCGGATGTCCTGGAG-3′; FL reverse, 5′-tgcatttcggcatTTAGCTTTGTTGA-3′ (lower case denotes region in pUAST vector). FL-dtau cDNA was then cloned into pENTR/D-TOPO entry and pDEST17 expression vectors, according to the manufacturers’ instructions (Invitrogen), and confirmed by sequencing. For dTau recombinant protein expression and subsequent antibody production, BL21-DE3 cells (Invitrogen) were transformed with mini-prepped (Qiagen) FL-dtau cDNA, and induced by incubation with 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside). Recombinant proteins were purified, using a Nickel-NTA column, by Fast protein liquid chromatography (AktaPurifier, GE Healthcare) across a 25–400 mM imidazole gradient. dTau-containing fractions were TCA-precipitated and gel-purified by SDS-PAGE, after which bands were excised and sent for antibody production (Eurogentech). Rabbit SK6427 anti-dTau antibody was used for subsequent experiments.