Heparin

The DNA encoding the full-length human XPA was cloned into a modified pET28a vector with a cleavable N-terminal His₆-SUMO tag for expression in E. coli Rosetta (DE3) pLysS cells (Invitrogen). After induction for 18 h with 0.2 mM IPTG at 22°C, the cells were harvested by centrifugation and the pellets were resuspended in lysis buffer containing 50 mM Tris–Cl pH 7.5, 500 mM NaCl, 10% glycerol. The cells were then lysed by sonication and the cell debris was removed by centrifugation. The supernatant was purified by Ni-NTA affinity chromatography and SUMO protease was then added to remove the His₆-SUMO tag. XPA was further purified with the Heparin (GE) and Superdex 200 (16/60, GE) columns. The purified XPA protein samples were concentrated in 25 mM Tris–Cl pH 7.5, 200 mM NaCl, 5% glycerol, 2 mM DTT, and stored at –80°C.

Recombinant heparin-assembled P301S Tau fibrils were prepared as described by KrishnaKumar and Gupta (2017) (link) with the following modifications: for protein purification we used the buffer A (50 mM MES pH 6,25, 0,5 mM DTT) and buffer B (50 mM MES pH 6,25, 0,5 mM DTT, 1 M NaCl) with the HiLoadTM 16/10 SP Sepharose^TM High Performance column (GE Healthcare Life Sciences); the protein was eluted using a linear gradient 0–100% of buffer B in six column volume; for protein aggregation 400 μg/ml heparin (Sigma Aldrich) has been added.
Cells were plated in glass bottom dishes as previously described and 1.2 μg of P301S Tau fibrils were delivered to cells with 2 μl of Lipofectamine 2000 transfection reagent diluted in 300 μl of Opti-MEM Reduced Serum Medium (Gibco). Cells were treated for 2 h, then DMEM low glucose, DMEM/F-12 or Neurobasal-A were added back to HeLa, SH-SY5Y or primary neurons, respectively. Scale up and scale down were performed as needed. The day after Tau seeding, cells have been treated with 1 μM PD-901 (sc-205427; Santa Cruz Biotechnology) or 6 μM D-JNKI-1 (HY-P0069/CS-5624; MedChemExpress) for 48 h in the presence of fibrils, while 10 μM STS (Cell signaling technology) or 200 nM OA (Cell signaling technology) were administered 72 h after Tau seeding for 1.5 h.

The catalytic fragment of Arabidopsis JMJ13 (JMJ13CD, residues 90–578) was cloned into a pET-Sumo vector to fuse an N-terminal hexahistidine plus yeast sumo tag. The plasmid was transformed into E. coli strain BL21(DE3) RIL and the transformants were cultured at 37 °C in LB medium. When the OD₆₀₀ of cell culture reached 0.7, the protein expression was induced by adding IPTG to a final concentration of 0.2 mM and the cells were cooled to 20 °C. The recombinant expressed protein was purified using a HisTrap column (GE Healthcare). The hexahistidine plus yeast sumo tag was removed by ulp1 protease digestion followed by a second step HisTrap column (GE Healthcare). The target protein was further purified on Heparin and Superdex G200 columns (GE Healthcare). The untagged JMJ13CD easily precipitates in the in vitro activity assay. We further cloned JMJ13CD into a pMal vector (New England Biolabs) to fuse an MBP tag to the target protein. The MBP-tagged JMJ13CD was expressed in E. coli strain BL21(DE3) RIL with IPTG induction and purified using amylose resin (New England Biolabs), Heparin, and Superdex G200 columns (GE Healthcare). All the mutations were generated using a PCR based method and purified using the same protocol as wild-type protein. The chemicals and peptides were purchased from Sigma-Aldrich and GL Biochem Company, respectively.