Phenylmethylsulphonyl fluoride pmsf

Human WT α-syn was prepared as described previously (Smith et al., 2008 (link)). Briefly, α-syn was expressed in Escherichiacoli BL21 (DE3) cells (New England Biolabs, Ipswich, MA, USA). Expression was induced using autoinduction media (Formedium, Norfolk, UK) at 37°C for 24 h. Bacterial cells were harvested by centrifugation at 4°C, 10,000 g and resuspended in lysis buffer [10 mM Tris-HCl (pH 8.0), 100 µg/ml lysozyme (Sigma-Aldrich, Poole, UK), 1 mM phenylmethylsulphonyl fluoride (PMSF; Sigma-Aldrich), 20 µg/ml DNase (Sigma-Aldrich), 20 µg/ml RNase (Sigma-Aldrich) and protease inhibitor cocktail (Life Technologies, Paisley, UK)]. The protein pellet was subjected to anion exchange followed by size exclusion chromatography. Lyophilised purified α-syn was stored at −20°C. Protein concentration was measured using a UV/vis spectrophotometer (CECIL 1000) at absorbance 280 nm with an extinction coefficient of 5960 M⁻¹ cm⁻¹.

In order to obtain protein extracts for Western blot analysis mESC were lysed with 100μl of RIPA buffer (Sigma-Aldrich) supplemented with 2mM of phenylmethylsulphonyl fluoride-PMSF (Sigma-Aldrich) and 2x Halt phosphatase inhibitor cocktail (Pierce, Rockford, IL) as described elsewhere [8 (link)]. Protein quantification was performed using the Pierce BCA (Bicinchoninic Acid) Protein Assay Kit, following the datasheet protocol. Both samples and calibration curve were determined with duplicates.

DMGT and CF41.Mg cells were grown in serum-free OPTI-MEM (Gibco, Thermo Fisher Scientific) for 24 h, and the media were collected and centrifuged at 5000 rpm for 5 min to remove detached cells. The media were subsequently concentrated and desalted with Vivaspin^® 6 sample concentrators (GE Healthcare). For cell lysate extraction, cells were washed with 1× PBS three times and lysed on ice with the lysis buffer (50 mM Tris-HCl (pH 8.0), 5 mM EGTA (Sigma-Aldrich, Merck, Burlington, MA, USA), 5 mM EDTA (Amresco-VWR, Radnor Township, PA, USA), 1% Triton X-100, 20 mM sodium pyrophosphate) freshly supplemented with a protease inhibitor cocktail (VWP Life Science, Avantor, Radnor Township, PA, USA) and phenyl methyl sulphonyl fluoride (PMSF; Sigma-Aldrich). Cell lysates were collected into a microcentrifuge tube and centrifuged at 12,000 rpm at 4 °C for 15 min, and the resulting supernatant was transferred to another microcentrifuge tube. Protein concentrations of the conditioned media and cell lysates were measured using a BCA protein concentration assay kit (Pierce, Thermo Fisher Scientific).

Cell culture media was removed, and infected or activated cells were washed twice with ice-cold PBS. Cells were collected by scraping into 5 mL of ice-cold PBS and harvested by centrifugation at 1000 g for 5 min at 4°C. The medium was completely aspirated, and cells were lysed for 10 min at 4°C by the addition of 200 µL of the extraction buffer (50 mM of HEPES pH 7.0, 250 mM of NaCl, 5 mM of EDTA, 0.1% Nonidet P-40, 50 mM of NaF, 0.5 mM of Na₃VO4, 1 mM of phenylmethylsulphonyl fluoride (PMSF) containing 5 μg/mL each of aprotinin, leupeptin, and pepstatin, all compounds from Sigma–Aldrich). Cell debris was removed by centrifugation at 1000 g followed by quick freezing of the supernatant. The protein concentration in the supernatant was determined using the Bio-Rad DC assay (Bio-Rad Laboratories), and 5 µg of protein was immediately used for Western blotting.

Murashige and Skoog medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, ethylenediaminetetraacetic acid (EDTA), triphenyl-tetrazolium chloride (TTC), 5-sulphosalicylic acid, 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), deferoxamine, dimethyl sulfoxide (DMSO), acrolein, α-Tocopherol, Glutathione Reductase (GR), glutathione (GSH) and phenylmethylsulphonyl fluoride (PMSF) were obtained from Sigma-Aldrich. Ferrostatin-1, RSL3, Z-VAD-FMK and liproxstatin-1 were purchased from Selleckchem. 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA) was purchased from Invitrogen. Ac-DEVD-AMC, Ac-DEVD-CHO and E64-d were from MedChemExpress. All other chemicals were from analytical or HPLC grade, and was purchased from Reanal, Hungary.

Seven days after the last oral vaccination, fresh fecal pellets were collected and added to the extraction buffer (0.01 M ice-cold PBS, pH=7.2, 0.5% Tween, and 0.05% sodium azide) at a ratio of 1 ml per 100 mg fecal pellets wet weight. Tubes were vortexed for 15 min at room temperature and the fecal suspensions were centrifuged at 3000×g for 15 min at 4°C. A portion of the supernatant (400 μl) was transferred to a sterile eppendorf tube containing100 μl glycerol to which 10 μl of Phenyl Methyl Sulphonyl Fluoride (PMSF, Sigma) solution was added and then vortexed briefly. Samples were centrifuged at 14,000×g for 15 min at 4°C. Supernatants were transferred to sterile tubes and stored at −20°C until use ^{14 (link)}.