Mini edta free protease inhibitor

After sacrifice, 24 h after the last TMA challenge, ear tissues of mice were collected and homogenized in a saline to a concentration of 100 mg/ml with a cOmplete, Mini, EDTA-Free Protease Inhibitor (Roche Applied Science), and the debris-free supernatant was used for cytokine measurement. The levels of IL-4, IL-5, IL-13, and in-ear homogenates were measured by ELISA kits (R&D Systems Inc., MN, USA) according to the manufacturer's protocol.

An E. coli codon-optimized coding sequence (GenScript) facilitates robust recombinant overexpression of human APTX^{24 (link)}. WT and H260N human APTX catalytic domain (cat, residues 165–342) were expressed from pET15b as N-terminal 6× His-tagged proteins in E.coli BL21(DE3) codon-plus cells (Novagen). Cell cultures were grown at 37 °C in LB medium containing ampicillin (100 μg mL⁻¹) and chloramphenicol (34 μg mL⁻¹) until the A₆₀₀ reached 0.8–1, at which time cells were cooled to 16 °C, and grown for an additional 8–12 h, without IPTG induction. Cells were lysed by sonication in lysis buffer (50 mM Tris, pH 8.5, 500 mM NaCl, 10 mM imidazole, 0.01 g/L lysozyme, with Roche mini EDTA-free protease inhibitor). The soluble lysate was applied to Ni-NTA column (5 mL, Qiagen) and 6× His-tagged APTX proteins were eluted in lysis buffer with 300 mM imidazole. The 6× His-tag was removed with overnight thrombin digestion (50U) (Sigma) at 4 °C. Subsequent purification was achieved by size exclusion chromatography (Superdex 75, GE healthcare in 50 mM Tris, pH 7.5, 500 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol) and cation exchange chromatography on a 5 mL Hi-Trap SP HP (GE Healthcare).

Protein extraction was performed using RIPA buffer in the presence of a complete Mini EDTA-free protease inhibitor (Roche) and PhosSTOPTM phosphatase inhibitor (Roche). Protein separation was performed by SDS-page using Bolt 4 to 12% Bis-Tris protein gels (Invitrogen). Transferences were done in an iBlot 2 gel transfer device using iBlot 2 transfer nitrocellulose stacks (Invitrogen). Membranes were blotted against phospho-AKT (Ser473) (Cell Signaling), phospho-RAPTOR (Ser792) (Cell Signaling), phospho-p70 S6 Kinase (Thr389) (Cell Signaling), β-ACTIN (Cell Signaling) and their correspondent HRP-conjugated secondary antibodies. The revelation was performed using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and images were acquired using an ImageQuant LAS 4000 (GE Healthcare).

After weighing and mashing, fresh liver tissues were lysed in PBS buffer supplemented with protease inhibitors (Mini EDTA-free Protease Inhibitor, Roche, Switzerland) and broken by sonication. After centrifugation (5,000×g, 10min), the supernatant was used for nerve growth factor (NGF) assays according to the manufacturer’s protocol (Jianglai Biotechnology Co., Shanghai, China). Absorbance at 450nm was measured using a microplate reader (Bio-Rad, Hercules, United States). Each measurement was performed in triplicate.

Tissue was homogenized in Radio-immunoprecipitation (RIPA) Buffer (Sigma-Aldrich R0278) with a complete Mini EDTA-free Protease Inhibitor (Roche). The protein concentration of the lysate was quantified by a BCA protein assay kit (Pierce). About 120–200 micrograms of total protein from the striatum were used for quantification of Tyrosine hydroxylase using a sandwich ELISA kit (Antibodies online-ABIN6960326) according to the manufacturer’s instructions.