Nα tosyl l lysine chloromethyl ketone hydrochloride

Adalimumab (Cyltezo^®, ADA) was kindly provided by Boehringer Ingelheim Pharma GmbH and Co. KG (Biberach, Germany). Poly-lactide-co-glycolide (Resomer^® RG 502 H, PLGA) was purchased from Evonik (Essen, Germany). 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), N-α-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), papain and polyvinyl alcohol (Mowiol^® 4-88, PVA) and lipopolysaccharide (LPS) from Salmonella enterica serotype abortus equi were purchased from Sigma Aldrich (St. Louis, MO, USA). Human TNF-α ELISA was purchased from Thermo Fisher (Waltham, MA, USA). Adalimumab ELISA was purchased from AffinityImmuno (Charlottetown, Canada). Mouse TNF-α and IL-1b ELISA were purchased from Merck (Darmstadt, Germany). All other chemicals and organic solvents were of analytical grade.

Fractionation of all cell lysates was performed by sequential extraction using buffers of increasing strength. Cells from 6-well culture dishes were scraped into 300 μl RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), a mixture of protease inhibitors (1 μM pepstatin, leupeptin, N-p-Tosyl-l-phenylalanine chloromethyl ketone, Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride, trypsin inhibitor; Sigma), and a mixture of phosphatase inhibitors (2 mM imidazole, 1 mM NaF, 1 mM sodium orthovanadate; Sigma). Samples were sonicated and centrifuged at 100,000xg for 30 min at 4°C, and then re-extracted in RIPA buffer to ensure complete removal of soluble proteins. Resultant insoluble pellets were extracted in 100 μl urea buffer (7 M urea, 2 M Thiourea, 4% CHAPS, 30 mM Tris, pH 8.5) containing the same formulation of inhibitors as described for RIPA buffer, then sonicated and centrifuged at 100,000xg for 30 min at room temperature. Soluble and insoluble fractions were analyzed by western blotting using the indicated antibodies. Tau antibodies used for western analysis were as follows: C-terminal T46 (1:1000, Thermofisher), tau-5 (1:1000, Thermofisher), tau-1 (1:1000, Thermofisher), K9JA (1:10,000, Dako), ac-K280 [3 (link)] (1:1000), and ac-K163 (1:1000, this study).

N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK, # T4376), N-α-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK, #90182), suberoylanilide hydroxamic acid (SAHA, #SML0061), anacardic acid (#A7236), decitabine (#A3656), lipopolysaccharide (LPS from Escherichia coli O111:B4, #L4391) were purchased from Sigma-Aldrich. Carfilzomib (#S2853) and tocilizumab (#A2012) were purchased from Selleckchem. Ro-106-9920 (#1178), TPCA-1 (#2559), PS1145 (#4569), Bay11-7082 (#1744), Ruxolinitib (#7048) and Stattic (#2798) were purchased from TOCRIS bioscience. TAK-242 (# 614316) was purchased from Millipore. Etanercept was supplied by Pfizer.

Commercial wheat gliadin (Sigma-Aldrich, St. Louis, MO, USA) was subjected to pepsin (0.1 M HCl, pH 1.8) (Sigma-Aldrich, St. Louis, MO, USA) and then trypsin (pH 7.8) (Sigma-Aldrich, St. Louis, MO, USA) digestion at 37 °C for 4 h with vigorous agitation. The pH was adjusted to 4.5 resulting in a precipitate, which was removed by centrifugation. N-tosyl-l-phenylalanine chlorome-thyl ketone and N- α-tosyl-l-lysine chloromethyl ketone hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) were used to inhibit the residual enzymatic activity. The solution was dialyzed, filtered and lyophilized. The powder was dissolved in sterile water at 50 mg/mL, aliquoted and stored at −80 °C.

For nuclease substrates, calf thymus DNA was obtained from Sigma-Aldrich (St. Louis, MO, United States) and baker’s yeast RNA was purchased from Fisher Scientific/Alfa Aesar (Haverhill, MA, United States). RNAlater stabilization solution was purchased from Invitrogen (Carlsbad, CA, United States). The protease substrates azocasein, N_α -Benzoyl-D,L-arginine 4-nitroanilide hydrochloride (BApNA), N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA), L-Leucine p-nitroanilide (LpNA), pGlu-Pro-Leu p-nitroanilide (pGFLpNA) were obtained from Sigma-Aldrich. Z-Arg-Arg p-nitroanilide (zRRpNA) was acquired from Bachem (Bubendorf, Switzerland). The inhibitors Phenylmethylsulfonyl fluoride (PMSF), N_α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), N_α -Tosyl-L-phenylalaninechloromethyl ketone (TPCK), E-64, Ethylenedintrilotetraecetic acid (EDTA) were also purchased from Sigma-Aldrich.

The digestion procedure was performed as described by Frazer et al. [58 (link)], with some modifications. Briefly, gliadin (Sigma-Aldrich, St Louis, MO, USA) was digested with pepsin (0.1 M HCl, pH 1.8) (Sigma-Aldrich, St Louis, MO, USA) and then trypsin (pH 7.8) (Sigma-Aldrich, St Louis, MO, USA) at 37 °C for 4 h with vigorous agitation. Adjustment of the pH to 4.5 resulted in a precipitate, which was removed by centrifugation. To inhibit the residual enzymatic activity, both N-tosyl-l-phenylalanine chloromethyl ketone and N- α-tosyl-l-lysine chloromethyl ketone hydrochloride (Sigma-Aldrich, St Louis, MO, USA) were used. After dialysis, PT was sterile filtered and lyophilized. The resulting powder was dissolved in sterile water and stored at −20° C.