Amicon ultra device

Conditioned medium was thawed, clarified by centrifugation at 25000 × g, and filtered through a 0.22 μm membrane (Steritop filter, Millipore, USA). The medium was diluted twofold with PBS, and the final pH was adjusted to 7.0, when necessary. IMAC purification was performed using cobalt-coated TALON beads (Clontech, USA) in batch mode using 2 l Erlenmeyer flasks. Following a 30-min incubation period with shaking at 110 rpm, the beads were collected in a gravity flow Econo column (Bio-Rad, USA), washed with PBS and the bound His-tagged protein was eluted with PBS containing 250 mM imidazole. The eluate was concentrated with Amicon Ultra device (10 kDa cut-off membrane, Millipore) and subjected to gel filtration on Superdex 200 HR 10/30 column (GE Healthcare, USA). All steps were performed at room temperature. Protein concentration was determined by Bradford assay (Bio-Rad). All pHLsec-FcHis constructs were cleaved with HRV 3C protease overnight, at 4 °C, in non-reducing conditions at a 1:5 target-to-protease mass ratio; all pYD5 constructs were captured on a Protein A column (MabSelect SuRe, GE Healthcare, USA), followed by cleavage with TEV protease and purification by gel filtration, as described above.

FTS/YM155 NCs and FTH1/YM155 NCs were prepared by urea‐mediated and pH‐mediated disassembly/assembly methods with minor modification, respectively.²¹ Briefly, YM155 dissolved in PBS solution (1 mM) was added to FTS NCs or FTH1 NCs dissolved in PBS solution (2 μM, 5 mL) at NCs/YM155 molar ratio of 1:200. Among them, FTS NCs from mixed solutions were disassembled into subunits by 8 M urea, stirred for 60 min, and sequentially dialyzed with Buffer C. FTH1 NCs from mixed solutions were adjusted to pH 2.0 by 0.1 M HCl to disassemble into subunits, and then increased to pH 7.4 with 1.0 M NaOH after stirring for 60 min, and dialyzed with Buffer D. After dialysis and centrifugation, the supernatants were concentrated with30 kDa Amicon Ultra device (ACS503002) (Millipore) and filtered by 0.22‐μm sterile filter (F513163‐0001) (Sangon Biotech). DOX‐loaded NCs and cyanine‐loaded NCs were fabricated in a similar way.

Freshly reduced AtAMY3, obtained as previously described, was incubated with 5 mM GSSG for 90 min at 25°C. After GSSG incubation, sample was desalted through NAP-5 column (GE Healthcare) in 100 mM Tricine-NaOH, pH 7.9 and brought to the desired concentration (20 μM) through Amicon-Ultra device (Millipore; cut-off 10 kDa). Sample concentration was determined by absorbance at 280 nm (Nanodrop; Thermo Fisher Scientific).
Reactivation treatments of glutathionylated AtAMY3 were performed by incubating samples for 30 min at 25°C in the presence of 50 μM, 0.2 mM, 1 mM, 2 mM, 5 mM, and 7 mM GSH. Samples were then diluted 5-fold and the activity assayed as described above.
Reactivation assays of GSSG-inhibited AtAMY3 performed in the presence of poplar GRX S12 and A. thaliana GRX C5, and commercially available E. coli TRX (Sigma-Aldrich; protein id AAA24693), were conducted through 5, 15, and 30 min of incubation of 20 μM glutathionylated AtAMY3 in the presence of 2 mM GSH with or without 5 μM GRXs, or in the presence of 0.2 mM DTT with or without 10 μM TRX. Upon incubation, enzyme activity was measured on 5-fold diluted samples as described above.
All data are expressed as percentages of activity relative to the fully reduced sample obtained by incubating glutathionylated AtAMY3 in presence of 80 mM DTT for 30 min at 25°C.

trypsin digestion and LC–MS/MS analysis were performed by KANEKA TECHNO RESEARCH. One hundred µg of Fc(aMD4ds)T1 or Fc(aMD4ds)B3 in PBS were treated with 100 mM ammonium bicarbonate (Sigma-Aldrich) containing 8 M urea (Fujifilm Wako Pure Chemicals). After desalting using an Amicon Ultra device (MWCO: 10 K, Millipore), 10 µg of each protein was digested with trypsin (Promega) for 12 h at 37 °C under non-reducing conditions. The reaction was stopped by the addition of formic acid. The samples were injected into a Nexera X2 UHPLC system (Shimadzu corporation) connected to a SCIEX TripleTOF 6600 system (AB SCIEX) equipped with an ESI ionization source. Peptides were chromatographically separated on a C18 reverse-phase column with linear-gradient of mobile phase A (water containing 0.1% formic acid) and mobile phase B (acetonitrile (Fujifilm Wako Pure Chemicals) containing 0.1% formic acid). MS and MS/MS data were collected using IDA, positive ion mode. The LC–MS raw data were processed using SCIEX BioPharmaView software. The following criteria were used to identify peptides: mass accuracy for the matched precursor must be within 5 ppm of mass error, and peptide matching score was set to at least 3 for peptide identification.

Unlabeled or ¹³C/¹⁵N-labeled human TFIIH p62 PH-D (residues 1–108) and unlabeled or ¹³C/¹⁵N-labeled human RPB6 (residues 1–127) were prepared as previously described (5 (link)). In brief, p62 or RPB6 was expressed as a hexa-histidine-tagged product in a pET15b vector (Merck Millipore) in Escherichia coli BL21 (DE3) Gold (Agilent Technologies). The lysed supernatant was loaded onto a Ni-nitrilotriacetic acid (NTA) column (QIAGEN), and the eluate was digested with thrombin to remove the histidine tag. After concentration with an Amicon Ultra device (Merck Millipore), the sample was purified on a Superdex75 column (GE Healthcare).

Prior to oxidation and subsequent conjugation via reductive amination, S. typhimurium OAg-negative GMMA were concentrated at 10 mg/mL and exchanged in 100 mM acetate buffer pH 4.5. GMMA were oxidized with NaIO₄ (15 mM in the final reaction mixture) for 30 min at 25 °C, and then, the reaction was quenched with Na₂SO₃ at a final concentration of 30 mM, mixing for 15 min at RT. Reductive amination, between the previously derivatized polysaccharide with a dihydrazide linker (i.e., ADH, [36 ]) and reactive aldehydes of oxidized sugar residues of GMMA lipopolysaccharide (LPS) core, was conducted ON at RT with GAC/GMMA/NaCNBH₃ w/w/w ratio of 3:1:1. GAC-GMMA conjugate was then purified through centrifugal ultrafiltration using Amicon Ultra device with a membrane cut-off of 100 kDa (Merck, Darmstadt, Germany), and removal of unconjugated polysaccharide was verified via HPLC-SEC analysis with respect to a calibration curve of purified unconjugated polysaccharide (TSK gel G3000 PWXL column, refractive index detector). Total protein quantification in GAC-GMMA conjugate was estimated by micro-BCA analysis, while GAC amount estimation was based on Rha quantification through HPAEC-PAD analysis [37 (link),38 (link)], as GlcNAc is also present in the LPS outer core of S. typhimurium OAg-negative GMMA. Conjugate size distribution was determined by DLS [39 (link)].

For secretome collection, MenSCs (n = 5) at passages P6–P9 were cultured in DMEM with 1% penicillin/streptomycin, 1% glutamine, and 1% insulin-transferrin-selenium (ITS, Thermo Fisher Scientific) for 48 h under N (N/S-MenSCs) and AH (AH/S-MenSCs) conditions. ITS replaced FBS to avoid external EV contamination. After that, conditioned media were collected and centrifuged, first at 1000×g for 10 min at 4 ºC and then at 5000×g for 20 min at 4 ºC to eliminate debris and dead cells. The supernatants were filtered through 0.45 µm and 0.22 µm meshes (Fisher Scientific, Leicestershire, UK) and concentrated by centrifugation at 4000×g for 40 min at 4 °C, using a 3 kDa MWCO Amicon® Ultra device (Merck-Millipore, MA, USA). Finally, protein concentration was measured by Bradford assay (Bio-Rad). The concentrated secretomes were stored at – 80 °C for further analyses.