Sodium deoxycholate

To separate nuclei from cytoplasm in CNs, an Abcam protocol was applied with slight modifications. In brief, cells were scraped off on ice in fractionation buffer [20 mM HEPES (pH 7.4) (Carl Roth), 10 mM KCl (Carl Roth), 2 mM MgCl₂ (Merck Millipore), 1 mM EDTA (Carl Roth), 1 mM EGTA (AppliChem), 1 mM dithiothreitol (AppliChem) and 1×PI] and incubated for 15 min on ice. Samples were then passed through a 27-gauge needle (Sigma-Aldrich) ten times, then incubated on ice for 20 min. Samples were centrifuged at 750 g for 5 min at 4°C and supernatant containing cytoplasm was transferred to a fresh tube. One quarter of the total amount of 5×RIPA buffer [750 mM NaCl (VWR), 5% Igepal CA-630 (Sigma-Aldrich), 2.5% Sodium deoxycholate (Carl Roth), 0.5% SDS (Sigma-Aldrich) and 250 mM Tris (pH 8.0) (AppliChem)] with 1×PI was added. The nuclear pellet was washed with fractionation buffer followed by ten more passes through a 27-gauge needle and 10 min centrifugation at 750 g at 4°C. The supernatant was discarded, the pellet was resuspended in 1×RIPA buffer plus PI and sonicated to shear genomic DNA. Protein isolation was performed according to the procedure described above.

Human ISG15^–/– dermal fibroblasts were seeded at a density of 0.4 × 10⁶ cells per well in 6-well plates, and the next day 2 μg each of empty vector (pcDNA 6.0) or vector containing ISG15-WT or ISG15-mutant (c.288C>G) cells was transfected using Lipofectamine LTX reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cells were harvested in RIPA buffer (50 mM Tris [pH 8.0; Thermo Fisher Scientific], 150 mM NaCl [Th. Geyer GmbH & Co.], 0.1% SDS [Merck], 0.5% sodium deoxycholate [Carl Roth], and 1% Nonidet P-40 [Sigma-Aldrich]) containing complete Mini Protease Inhibitor Cocktail (Roche) and PhosSTOP tablets (Sigma-Aldrich) for immunoblots.