Protease inhibitor mixture

For immunoprecipitation, 293T cells at a confluency of 60%–70% in six‐well plates were transfected with a total of 5 μg plasmids that code for the protein of interest. Twenty‐four hours after transfection, cells were washed once with PBS and lysed in buffer A containing 25 mM Tris‐HCl, pH 7.5, 150 mM NaCl, 10% glycerol, and 1% Triton X‐100, supplemented with a protease inhibitor mixture (Roche Molecular Biochemicals). Precleared lysates were subjected to anti‐Flag M2 or anti‐GFP immunoprecipitation following the manufacturer's instructions. The beads were washed four times with lysis buffer, and the immunoprecipitates were eluted with sodium dodecyl sulfate (SDS) sample buffer, followed by standard immunoblotting analysis. All the immunoprecipitation assays were performed more than three times, and representative results are shown. For enrichment of the arginine‐GlcNAcylated proteins from lysates of transfected cells, samples were washed three times in ice‐cold PBS and lysed in buffer A containing 25 mM Tris‐HCl, pH 7.5, 150 mM NaCl, 10% glycerol, and 1% Triton X‐100, supplemented with a protease inhibitor mixture (Roche Molecular Biochemicals). Precleared lysates were subjected to immunoprecipitation with the anti‐Arg‐GlcNAc antibodies
²⁹ . The beads were washed four times with the lysis buffer, and the immunoprecipitates were dissolved by SDS sample buffer.

The left hemispheres of the mice from four groups were homogenized in ice-cold PBS containing 5 M guanidine hydrochloric acid and protease inhibitor mixture (Roche Diagnostics). The femurs extracts were prepared by extracting frozen pulverized bone tissue and suspending in ice-cold PBS containing 5 M guanidine hydrochloric acid and protease inhibitor mixture (Roche Diagnostics). Total protein content in the brain hemispheres and femurs extracts was determined via colorimetric BCA assay in accordance with the manufacturer’s recommendations (Jiancheng, Nanjing, China). The levels of Aβ42 and Aβ40 in the brain hemispheres and femurs extracts were quantified using ELISA kits (Invitrogen, Camarillo, CA, USA).
Blood samples were obtained from posterior-orbital venous plexus, centrifuged at 5000 r/min for 15 min at 4°C, and then stored at −80°C for late use. The plasma was collected and stored in -80°C. Before analysis, the plasma samples were thawed to room temperature. The serum concentrations of TRACP 5b, IL-6 and TNF-α were assayed using ELISA kits (Jian Cheng, Nanjing, China). Assays were performed in accordance with the manufacturer’s recommendations.

Brain samples were collected from Spraque Dawely rats, suspended in lysis buffer (50 mM Tris- HCl, pH 7.5, and 150 mM NaCl) containing protease inhibitor mixture (Roche). Tissue samples were homogenized and sonicated. Cell membrane fractions were separated by centrifugation at 100,000 × g and cell pellet was resuspended in lysis buffer containing 1.5% IGEPAL and protease inhibitor mixture (Roche). Cell were rocked at 4 °C for one hour then supernatant was cleared by centrifugation at 16,000 × g for 20 min at 4 °C. Supernatant precleared with Pierce protein A/G magnetic beads (Thermo-Fisher) followed by incubation with primary antibody conjugated protein A/G magnetic beads overnight at 4 °C. Beads were washed and protein was eluted in SDS sample buffer (containing 10% SDS and 9.3% DTT) at 95 °C for 5 min. Samples were run on western blot as described above.

GAPDH (EC 1.2.1.13) activity was measured using the KDalert® kit (Thermo Fisher Scientific, Hemel Hempstead, UK) at 22 °C. Cells were resuspended in lysis buffer at 10⁷ cells ml⁻¹, and tumors were freeze-clamped and extracted at 50 mg ml⁻¹. G6PDH (EC 1.1.1.49) activity was assayed at 22 °C, as described (56 (link)). Tumors were extracted at 200 mg ml⁻¹, and cells were extracted at 10⁷ cells ml⁻¹ in 50 mm HEPES buffer containing 100 mm KCl, 10 mm phosphate buffer, 10 mm MgCl₂⁻, 1 mm dithiothreitol, and protease inhibitor mixture (Roche Applied Science) in a Precellys24 homogenizer coupled to a Cryolys® cooler (Stretton Scientific) at 4 °C. Protein content was measured using Direct Detect® (Millipore, Billerica, MA). Grx activity was measured using a 2-hydroxyethyl disulfide coupled assay (57 (link)). TrxR activity was measured in a linked reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) and GST (EC 2.5.1.18) activity with 2,4-dinitrochlorobenzene using assay kits (Sigma-Aldrich). For Grx, TrxR, and GST assays, tumor and cell samples were extracted with 50 mm potassium phosphate buffer, pH 7, containing 0.5 mm EDTA and protease inhibitor mixture (Roche Applied Science) in a Precellys24 homogenizer coupled to a Cryolys® cooler (Stretton Scientific) at 4 °C. Protein content was measured using Bradford reagent (Bio-Rad, Hemel Hempstead, UK).

Ganglia were partially thawed, the outer fascia removed by dissection, and a portion macerated with a scalpel before partial homogenization in either radioimmune precipitation assay buffer with protease inhibitor mixture (Roche) for QFWB (see below) or iTRAQ extraction buffer containing 6 m Urea, 2 m thiourea, 2% CHAPS, 0.5% SDS, and protease inhibitor mixture (Roche) for proteomic processing (see below). Samples were pooled by condition and manually homogenized in a dounce glass homogenizer. Homogenized samples were sonicated in a cup style sonicator six times for 15 s at power level 7.5 with vortexing for 30 s between each round of sonication. Samples were left on ice for 10 min before being revortexed then centrifuged at 20,000 g for 30 min at 4 °C. The resulting pellet containing proteins insoluble when processed in this manner was stored at −80 °C, and the supernatant was transferred to a fresh 1.5 ml tube to be processed for iTRAQ labeling as previously described (17 (link)–20 (link, link, link)).

Cell lysates were prepared using the ProteoJET Mammalian Cell lysis reagent (Fermentas) and the 1× protease inhibitor mixture (Roche Molecular Biochemicals, Pleasanton, CA). Immunoblotting was performed as described earlier [30] (link). Monoclonal antibodies against Bcl-2, GAPDH and cleaved caspase-9 (Cell Signalling, Boston, MA, USA) were used.