Protease inhibitor

MiDAC was purified from 1.25 l of adherent Flp-In 293 T-REx (R78007, Thermo Fisher Scientific) cell lines stably transformed with the Flp-In expression vector carrying FLAG-ELMSAN1/MIDEAS. The cells were grown in DMEM media (Gibco) supplemented with 10% FBS, 100 µg ml⁻¹ hygromycin and induced for 24 h with 1 µg ml⁻¹ doxycycline (Thermo Fisher Scientific). Cells were harvested and lysed using the classical Dignam protocol^{53 (link)}. The complex was isolated from the nuclear fraction using anti-FLAG M2 beads from Sigma-Aldrich. The nuclear fraction was mixed with washed FLAG M2 beads and incubated overnight at 4 °C. The next day, the beads were washed with wash buffer (20 mM HEPES (pH 7.9), 300 mM NaCl, 1.5 mM MgCl₂, 10% glycerol, 0.5 mM DTT and protease inhibitors (Sigma)) four times. The complex was eluted from the beads in the elution buffer (20 mM HEPES (pH 7.9), 100 mM NaCl, 1.5 mM MgCl₂, 0.5 mM DTT and protease inhibitors (Sigma)) after 30 min of incubation at 4 °C. This complex was flash frozen in liquid nitrogen and stored at −80 °C.

Frozen cells collected by trypsinisation from 4 x 15 cm petri were defrosted for 30 min on ice in LSB buffer (10 mM Tris-HCl pH 7.6, 1.5 mM MgCl₂, 20 mM 2-chloroacetamide, protease inhibitors (Sigma), 1 mM Na₂MoO₄, 1 mM Na₃VO₄ and 4 mM sodium tartrate). Samples were briefly vortexed and nuclei pelleted. The nuclear pellets were washed with LSB buffer and pelleted again. The nuclei were resuspended in Extraction buffer (50 mM Tris-HCl pH 7.6, 1.5 mM MgCl₂, 420 mM NaCl, 420 mM EDTA, protease inhibitors (Sigma), 1 mM Na₂MoO₄, 1 mM Na₃VO₄ and 4 mM sodium tartrate) and sonicated for 5 seconds. Flag-agarose slurry (Sigma) were prewashed 4 times with Extraction buffer and added to the protein samples. Volumes were adjusted to 1 mL with Extraction buffer. Samples were mixed under rotation for 2 h at 4°C. The beads were washed 3 times with ice cold Extraction buffer followed by a wash with 1 mL of PBS. Immune complexes were eluted with three sequential incubations with Elution buffer (6 % ammonium hydroxide in water, pH 11–13). Eluates were combined and lyophilized to dryness in a speed vacuum. For LC-MS/MS, samples were reconstituted in 25 μL of 0.2% formic acid in water and injected as indicated above. For western blot analysis, samples were resuspended in 100 μL of Laemmli sample buffer (60 mM Tris-HCl pH 6.8, 2 % SDS and 10 % glycerol) and 20 μL were loaded on a 4–12% SDS-PAGE.

HEK293T cells were transfected with DNAs coding for the indicated proteins using calcium phosphate at a 1:1:0.2 ratio for NEDD4-, IFITM3- and NDFIP2-specific DNA. Two days afterwards, the cells were lysed in 150 μL of lysis/IP buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA), supplemented with a cocktail of protease inhibitors (Sigma, cat. 4693132001). Immunoprecipitations were carried out for two–three hours using 1 μg of the monoclonal antibodies indicated above, followed by an additional two hours incubation time with Protein A–sepharose beads (GE healthcare, cat. 17-5238-01). After extensive washing, samples were analyzed using WB. IP and washing steps were carried out using the same lysis/IP buffer mentioned above. For WB analyses, the cells were instead lysed directly in 100 μL of RIPA buffer (150mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 50 mM TrisCl pH 8.0), supplemented with a cocktail of protease inhibitors (Sigma, cat. 4693132001), twenty-four hours after transfection. For ubiquitin experiments, DNA coding for HA-ubiquitin was added along with DNAs coding for IFITM3 or NDFIP2s at a ratio of 2:1:2, respectively. Cell lysis and IPs were then carried out as described above.

Protein extracts from cells were prepared using a modified Laemmli buffer (5% sodium dodecyl sulfate, 10% glycerol, 32.9 mM Tris-HCl pH 6.8) supplemented with protease inhibitors (Sigma–Aldrich). Brain tissues were prepared with a lysis buffer (Hepes 10 mM pH 7.9; NaCl 0.4 M, EGTA 0.1 M; glycerol 5%, dithiothreitol [DTT] 1 mM, PMSF 1 mM, protease inhibitor [Sigma–Aldrich], phosphatase inhibitor [Roche]). Then, 30 μg of proteins from lysates were subjected to migration on 8–12% acrylamide gels and transferred on to polyvinylidene difluoride membranes (GE Healthcare Europe GmbH) in borate buffer (50 mM Tris-HCl and 50 mM borate) for 1 h 45 at constant voltage (48 V). The membranes were incubated with primary antibodies overnight at 4 °C, then washed in Tris-buffered saline–Tween 0.1% and incubated for 1 h with horseradish peroxidase (HRP)-coupled secondary antibody (Jackson Immunoresearch). The signal was revealed using a chemiluminescent reagent (Pierce® ECL Plus Western Blotting Substrate, Thermo Scientific) and was detected using hyperfilm (HyperfilmTM ECL, Amersham Biosciences) and a film processor (Konica Minolta). Poly-ubiquitinated HSF2 was detected as described in ref. ^{21 (link)}.

Yeast cells grown in log phase were subjected to spheroplasting by supplementing the growth medium with 0.6 M sorbitol, 25 mM Tris-HCl (pH 7.5), 10 mM DTT, and purified lytic β-1,3-glucanase. Spheroplasts were washed with a buffer containing 0.4 M sorbitol, 20 mM PIPES–KOH (pH 6.6), 150 mM potassium acetate, 2 mM magnesium acetate, 1× protease inhibitors (Sigma), and lysed by a 5-min incubation on ice in an extraction buffer containing 20 mM PIPES–KOH (pH 6.6), 150 mM potassium acetate, 2 mM magnesium acetate, 1 mM sodium fluoride, 0.5 mM Na₃VO₄, 1× protease inhibitors (Sigma) and 1% Triton X-100. Chromatin sedimentation was carried out with 15-min centrifugation at 16,000 × g on a sucrose cushion (the extraction buffer supplemented with 30% sucrose). The pellets were washed and resuspended with the extraction buffer. Equal amounts of the whole-cell extract, non-chromatin, and chromatin fractions were analyzed with western blot with the anti-Pgk1 (22C5D8, Invitrogen, 1:8000 diluted), anti-histone H3 (ab46765, Abcam, 1:2000 diluted), and anti-TAP (P1291, Sigma-Aldrich, 1:12,000 diluted) antibodies.