Pierce protease inhibitor cocktail

The NIL was separated from the anterior lobe of the pituitary by blunt dissection and frozen in a 0.5 ml tube within 90 s of decapitation on dry ice. Total proteins were extracted in RIPA buffer (50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 0.1% sodium dodecyl sulfate; 0.5% sodium deoxycholate; 1% Nonidet P-40; 1 mM EDTA) supplemented with 1 mM PMSF, pierce protease inhibitor cocktail (Thermo Fisher Scientific, A32963) and phosphatase inhibitor cocktail (Thermo Fisher Scientific, A32957). Lysis buffer (80 μl/sample) was added and samples which were immediately sonicated in tubes kept on iced water for 12 s of sonication (MSE Soniprep 150). Samples were sonicated for 3 rounds of 12 s with intervals on ice of approximately 5 min. Samples we maintained on ice for an additional 30 min, vortexing every 5 min. To remove cellular debris samples were centrifuged at 10000×g for 20 min at 4 °C. The supernatant was removed and stored at −80 °C. Protein concentrations were determined in triplicate by Bradford assay with BSA standards using an iMark microplate absorbance reader (Bio-Rad Laboratories).

After sacrifice animals were lavaged with 1 ml PBS containing the Pierce protease inhibitor cocktail (ThermoFisher Scientific) and 1 mM EDTA and total cell counts were made.

Algae cultures were grown in a semi-continuous fashion in 1 L baffled flasks in TAP media wherein 95% of the culture was harvested via centrifuge and replaced with new media every two days; harvested call pellets were snap frozen until lysis. Cells were lysed in 1X Bugbuster (diluted from 10X buffer free stock) buffered with 50 mM Tris-HCl pH8.5 containing 1X Pierce Protease Inhibitor Cocktail (Cat#A32955; Thermo Scientific, Waltham, MA), 250 U/mL of Basemuncher endonuclease (ab270049, Abcam, Cambridge, MA), 1 mM DTT, 1 mM EDTA. 10 mL of lysis buffer was added to each gram of snap frozen wet biomass with typically 3–6 grams of wet biomass being processed at once. To the partially clarified cell lysate 10% wt/vol Polyethylenimine (Cat#408719, Sigma-Aldrich, St. Louis, MO), adjusted to pH 8.5 by addition of concentrated HCl was added to a final concentration 0.1% wt/vol. The lysate was then centrifuged at 6000 rcf at 4°C for 5 minutes and supernatant recovered. The lysate was then further decolorized by gently shaking against an equal volume of Methyl tert-Butyl Ether (MTBE) twice and then Hexanes (Fisher Scientific). The aqueous layer was then recovered, filtered through a 0.45 μm PES syringe filter and applied to a Capto Q anion exchange resin for column chromatography purification on an Akta pure 150 system fitted with an external sample pump (Cytiva, Amersham, UK).

Eluents from the ABE assay were dried for approximately 30 min and digested using the S-trap Micro Spin Column Digestion Protocol (Protifi, Huntington, NY) with minor changes. Briefly, 30 μL of 10% sodium dodecyl sulfate (SDS) and 100 mM triethylammonium bicarbonate (TEAB) with Pierce protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA) and phosphatase inhibitors (10 mM sodium pyrophosphate, 1 mM PMSF, 1 mM sodium orthovanidate, and 1 mM β-glycerolphosphate). Proteins were reduced with a final concentration of 20 mM dithiothreitol (DDT) at 95°C for 10 min, followed by alkylation in the dark at room temperature with 40 mM of iodoacetamide. Next, phosphoric acid was added for a final concentration of 1.2%. Samples were briefly vortexed to mix before 300 μL of S-trap binding buffer (90% MeOH, 100 mM TEAB) was added. Samples were vortexed again prior to loading onto the S-Trap Micro Spin Columns. After four washes with 150 μL of S-trap binding buffer with centrifugation at 1,000×g, 40 μL of 50 mM TEAB containing 0.75 μg of trypsin was added and incubated overnight at 37°C.
Peptides were eluted with 40 μL of each of the following solutions: 50 mM TEAB, 0.2% formic acid (FA), 50% acetonitrile (ACN), and 0.1% FA. The spin column was spun at 4,000×g after adding each solution. Pooled eluents were dried down prior to resuspension in 100 μL of 3% ACN and 0.1% FA.

Briefly, media conditioned with ActE flag-tagged at the N-terminus of the mature ligand was incubated with Anti-DYKDDDDK Affinity Resin (ThermoFisher, Cat No. A36801) at 4°C with gentle rocking. The resin was washed three times with 20 mM HEPES, 150 mM NaCl, and 1 mM ETDA. Subsequently, ActE was eluted from the resin with 100 mM Glycine pH 2.0, 150 mM NaCl, 0.5% CHAPS with Pierce™ protease inhibitor cocktail, EDTA free (ThermoFisher, Prod # 88266). Finally, fractions were pooled, concentrated, and dialyzed into 10 mM HCl.

Cell lysates post-treatment were prepared using ProteoJET Cell Lysis Reagent (Fermentas, Vilnius, Lithuania) supplemented with Pierce Protease Inhibitor Cocktail (Thermo Scientific, Hemel Hempstead, UK) and 10 mM Sodium Fluoride and 20 mM Sodium Orthovanadate (Sigma-Aldrich). Lysates were then denatured at 70°C for 10 minutes in Bolt^™ LDS-based loading buffer supplemented with DTT (Life Technologies, Paisley, UK). The denatured samples were run on Bolt^™ 12% Bis-Tris gels (Life Technologies) and transferred using standard Towbin Western transfer to 0.2 μm Immobilon PVDF membranes (Millipore, Watford, UK). Antibodies were used at 1:1000: Cleaved Caspase 3 (Cell Signaling Technology, Leiden, Netherlands), Caspase 3 (CST), PARP (C-2–10, Enzo Life Sciences, Exeter, UK) & β-actin (A5441, Sigma-Aldrich). Appropriate secondary antibodies (Dako, Ely, UK) were used at half the primary antibody concentration and developed using WesternBright ECL Reagent (Advansta, Menlo Park, USA).

Filter trap assays were performed essentially as described [96 (link)]. Briefly, HEK293T cells were co-transfected with EGFP-tagged poly-GA and either V5-tagged chaperones or the mCherry control plasmid as described above. After a 24 h period of transfection, cells were lysed on ice in Triton X-100 lysis buffer (1% Triton X-100, 15 mM MgCl₂ in PBS), supplemented with Pierce Protease Inhibitor cocktail (Thermo Fisher Scientific, A32965) and 30 units of DNase I. Equal amounts of lysates were centrifuged at 21,000×g at 4 °C for 30 min and insoluble pellets were resuspended in SDS lysis buffer (2% SDS in 100 mM Tris, pH 7.5) for 2 h at RT. Samples were diluted at 1:5 in SDS lysis buffer and filtered through a cellulose acetate membrane (0.2 μm pore size) using the Bio-Dot SF Microfiltration System (BioRad). The membrane was blocked in Intercept Blocking Buffer for 1 h and the protein bands were detected with antibodies against GFP (1:1000, Takara Bio, 632592), followed by Alexa Fluor Plus 800-conjugated secondary antibody (1:10,000, Invitrogen, A32735), and scanned on an Odyssey CLx imaging system (LI-COR).