Bl21 codonplus de3 rp

E. coli strain XL1-Blue (Stratagene, La Jolla, California) was used to grow and purify plasmids. BL21-CodonPlus-RP (DE3) (Agilent Technologies) and Rosetta (DE3) Competent Cells—(Novagen) were used for protein expression.

DF-1 cells^{32 (link)} were grown in monolayers in medium D-MEM supplemented with 5% foetal bovine serum (FBS), 2% of 4 mM L-glutamine and 1% antibiotic mix (penicillin and streptomycin). Sf9 cells (Thermo Fisher Scientific) were grown in suspension in medium SF-900II supplemented with 5% foetal bovine serum (FBS), 2% of 4 mM L-glutamine and 1% antibiotic mix (penicillin and streptomycin). E. coli strain XL1-Blue (Stratagene, La Jolla, California) was used to grow and purify plasmids. BL21-CodonPlus-RP (DE3) (Agilent Technologies) was used for protein expression.
Rabbit polyclonal antiserum against avian reovirus S1133 muNS protein was raised in our laboratory^{4 (link)}. Monoclonal antibody specific for SV5 Tag was obtained from Life Technologies. The following secondary antibodies were used as appropriate for different experiments: Alexa Fluor 594 conjugated antibodies against mouse or rabbit IgG; Alexa Fluor 488 conjugated antibodies against rabbit IgG (Invitrogen, Barcelona, Spain). Peroxidase-conjugated goat anti-rabbit IgG antibodies (Sigma-Aldrich, Madrid, Spain) were used for Western-blot analysis.

Escherichia coli BL21-CodonPlus (DE3)-RP (Agilent) was transformed with a pET28a plasmid encoding WT or P301L tau (full-length 0N4R). Terrific broth cultures (1 l) supplemented with 50 mg/l kanamycin or 50 mg/l chloramphenicol were inoculated with 20 ml of starter cultures and grown for 8 h. The cultures were induced with 1 mM IPTG and grown for another 16 h. Cells were harvested and resuspended in 50 ml/l 20 mM MES, pH 6.8, 1 mM EGTA, 1 mM magnesium chloride, 5 mM DTT, and 1 cOmplete protease inhibitor cocktail (Roche) followed by microfluidizer lysis. The lysates were boiled for 20 min and centrifuged at 48,400g. The cleared lysates were applied to a cation exchange column (SP Sepharose Fast Flow; GE Healthcare), and fractions were eluted with a sodium chloride gradient. Fractions containing 0N4R tau were applied to a reversed-phase HPLC column and eluted with an acetonitrile gradient (1%/min) + 0.1% TFA gradient; the peak fractions were then lyophilized. The lyophilizates were dissolved in PBS + 1 mM DTT and purified by size-exclusion chromatography (HiLoad 26/600 Superdex 200 pg; GE Healthcare). Peak fractions were analyzed by SDS-PAGE, and fractions containing <95% 0N4R tau were pooled, snap-frozen, and stored at −80 °C. Recombinant full-length α-synuclein and tau repeat domain (K18) were expressed and purified as previously described (72 (link), 73 (link)).