Pierce protease inhibitor mini tablet

EnzCheck^® Caspase-3 Assay Kit, TMRM, 5-(and-6)-carboxy-2′, 7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA), Pierce ^TM Protease Inhibitor Mini Tablets and Pierce^TM Phosphatase Inhibitor Mini Tablets were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Annexin V-FITC Apoptosis Detection Kit was obtained from Immunostep (Salamanca, Spain). Rap, Comp C and Baf A1 were obtained from Abcam (Cambridge, UK). Other chemicals were reagent grade and were purchased from Sigma-Aldrich (Madrid, Spain).

Animals at different developmental stages were anesthetized and decapitated for brain extraction. Brains slices of 300 μm were prepared on a vibratome (Leica VT1000S) in RNase-free 0.01 M PBS solution. Dorsal striatum was dissected for RNA extraction. For qPCR and Western blotting, RNA and proteins were isolated simultaneously by guanidinium thiocyanate-phenol-chloroform (AGPC) method using QIAzol lysis reagent (Qiagen, Germany). Briefly, samples were homogenized in QIAzol and the RNA was separated to an upper phase whilst the proteins remained in the lower phase. The upper phase was collected and further decontaminated by a second chloroform step. Subsequently, the RNA and proteins were precipitated separately and washed according to the QIAzol protocol. The quality and quantity of the RNA was accessed using the NanoDrop 1000 (Thermo Fisher Scientific). Synthesis of cDNA was performed by High Capacity cDNA Reverse Transcription Kit (Applied Biosystems^TM, Thermo Fisher Scientific, Belgium). The protein pellet was dissolved in 100 μl 1% sodium dodecyl sulphate (SDS) supplemented with Pierce^TM Protease Inhibitor Mini Tablets (Thermo Fisher Scientific) and stored at -20°C. The protein concentration was determined by bicinchoninic acid assay (Pierce BCA protein assay kit, Thermo Fisher Scientific).

At the experimental endpoint, medium was removed and N27 cells were washed two times with ice-cold PBS. Cells were collected by scraping the cells with non-pyrogenic sterile cell scraper (Corning Costar) in 300 μL ice-cold PBS. The collected cells were centrifuged for 5 min at 4 °C, 5 × 1000 g using Biofuge fresco, Heraeus, UK. PBS was removed and the cell pellet was suspended in 70 μL RIPA buffer (50 mM Tris- HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate, Sigma) supplemented with PierceTM Protease Inhibitor Mini Tablets (Thermoscientific) which contain protease- inhibitor cocktail (AEBSF, Aprotinin, Bestatin, E64, EDTA, Leupeptin, Pepstatin A), for each 10 ml of RIPA buffer, one 30 mg tablet of protease inhibitor was dissolved. After 30 min incubation of the cells in RIPA buffer supplemented with the protease inhibitor in ice, the lysate was centrifuged for 10 min at 4 °C, 5 × 1000 g using Biofuge fresco, Heraeus, UK. The supernatant was removed and stored at −20 for later protein quantification by western blot analysis, as described above for human samples. Supplementary Table 2 summarizes the primary and secondary antibodies, blocking and incubation conditions applied for western blotting in the cell culture experiments.

Log phase yeast cells were harvested (unstressed and after 2 h of treatment with 0.8 mM H₂O₂), and total protein was extracted using the acid-washed glass bead method to obtain cell lysate in 1× ice-cold passive lysis buffer (PLB) (DLR Kit, Promega), supplemented with Pierce Protease Inhibitor Mini Tablets (Thermo Fisher Scientific). LAR II and Stop-and-Glo® Reagent were prepared using the DLR Kit and DLR assay was performed according to the manufacturer's instructions (Promega) using a GloMax 20/20 Luminometer (Promega). The measurements for each strain were taken in triplicate. Statistical analysis was done in GraphPad Prism (GraphPad Software). For each strain the ratio Fluc/Rluc was used to calculate the %readthrough for each stop codon reporter UAA, UAG and UGA normalised to the CAA codon reporter (46 (link)). Similar analyses were carried out to calculate % frameshift for all strains with the –1 and +1 reporter plasmids (47 (link)). DLR ratios were normalised to the ratio of the frame 0 reporter for the strain and condition.

DH5ɑZ1 containing the pZA31:LACS4 construct was grown with 25 µg mL⁻¹ chloramphenicol at 37°C to an OD₆₀₀ of 0.5. LACS4 expression was induced with 200 µg mL⁻¹ anhydrotetracycline, then grown overnight at 16°C. Cells were pelleted, resuspended in 50 mM Tris pH 7.5, 300 mM NaCl, 0.1% Triton X-100 (v/v), and Pierce Protease Inhibitor Mini Tablets (Thermo Fisher). Cells were lysed by incubating with 2 mg/mL lysozyme at 4°C for 1 h followed by sonication with 10 30-s pulses. Cell debris was removed by centrifuging for 30 min at 20,000g at 4°C. Supernatant was then passed through a 0.22-µM filter to remove remaining debris.
The filtered supernatant was passed through an equilibrated 5-mL cobalt column on an ÄKTA prime FPLC (GE Healthcare) to capture HN-tagged LACS4. LACS4 recombinant protein was eluted in 1-mL fractions with 50 mM Tris pH 7.5, 300 mM NaCl, with increasing imidazole gradient up to 200 mM. Eluted fractions containing LACS4 were pooled and dialysed at 4°C in 10 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, and 40% glycerol (v/v). Protein concentration was determined by Bradford assay. LACS4 was aliquoted and stored at –80°C.