Nups proteins were a kind gift of S. Frey and D. Görlich. We used a maleimide-cysteine coupling reaction to conjugate the proteins with an oligonucleotide at the single cysteine at the N-terminal tails of both protein variants. The maleimide-modified oligo was produced by Biomers, Ulm, Germany. The proteins were treated with TCEP prior to incubation with the modified oligonucleotides which was subsequently removed using cut off filters (Merck Millipore). The proteins were incubated with the oligonucleotides in the presence of 5 M GuHCl-PBS overnight. The protein-oligo mixtures were purified from non-attached oligos by size exclusion fast protein liquid chromatography (AKTA) with Superdex 200 increase columns using the same buffer. First, control samples were analyzed using AKTA. One sample contained only the oligo with a malemide modification (’M-oligo’) and one sample contained only NSP1 proteins, to identify the elution peak for each sample. It is important to mention that the use of GuHCl in the running buffer was essential for this step. GuHCl ensures that NSP1 proteins remain in their unfolded conformation and do not interact with each other, forming aggregates which would result in clogging of the SEC column. The protein was stored at −80 °C until it was incubated with the rings.
Cut off filters
Manufactured by Merck Group
Sourced in Germany
Cut-off filters are laboratory equipment used to separate molecules or particles based on their size or molecular weight. These filters selectively allow the passage of molecules smaller than a specified size while retaining larger molecules or particles. The core function of cut-off filters is to purify, concentrate, or fractionate samples in various research and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Fluostar omega spectrophotometer, by BMG Labtech (1 mentions)
Fastprep, by MP Biomedicals (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Optima xpn 80, by Beckman Coulter (1 mentions)
Sw41ti, by Beckman Coulter (1 mentions)
Sw32ti, by Beckman Coulter (1 mentions)
6 protocols using cut off filters
1
Labeling Nups Proteins with Oligonucleotides
2
Measuring Brain Nitrosothiols in Mice
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Brain samples from PBS perfused mice were flash-frozen in dry ice and stored at −80 °C. Nitrosothiols were measured using a Nitrosothiols kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s instructions after filtration of samples through a 10,000 kDa cut-off filters (Merck Millipore, Darmstadt, Germany). Samples were read at 540 nm using a FLUOstar Omega spectrophotometer (BMG Labtech, Ortenberg, Germany).
3
Characterizing Active Compound in Conditioned Media
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To characterize the nature of the active compound conditioned media were exposed twice to high temperature (100 °C for 10 min) and subsequently cooled down on ice. Native conditioned media was size-fractionated using 100 kDa, 50 kDa, 30 kDa, 10 kDa, 3 kDa and 1 kDa cut-off filters (Millipore) whereby the flow-through was harvested and applied to the next smaller filter. All preparations were used for PPARγ activation testing as described below.
4
Characterization of Streptococcus salivarius Secretions
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The Streptococcus salivarius strains used in this study are listed in S1 Table . Bacteria were grown overnight at 37°C, S. salivarius, S. vestibularis and S. agalactiae in chemically defined medium (CDM) [39 (link)] and E. coli in Luriani Broth (LB). Bacterial cultures supernatants were collected after centrifugation and 0.22μm filtration. CDM adjusted to bacterial supernatant pH (pH≈5.5) was used as control. When mentioned, bacterial supernatants were size-fractionated using 10kDa and 3kDa cut-off filters (Millipore) or exposed to high temperature (100°C for 10 min) and heat shock assay (100°C/10 min prior to liquid nitrogen-freezing). Bacterial lysates were prepared by mechanical lysis using a FastPrep instrument (MP Biomedicals). 3kDa filtered bacterial supernatants and CDM were treated with proteinase K (100μg/ml, Sigma), DNase I (100μg/ml, Sigma) or trypsin (0,25%) for 2h at 37°C. Then, based on their molecular weight characteristics ranging from 23 to 29 kDa, the enzymes were eliminated by using a 10kDa cut-off filters.
5
Preparation and Use of α-Syn-Enriched Conditioned Media
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To prepare α‐Syn‐enriched conditioned media (α‐Syn‐CM), PC12 cells were infected with pLentiVENUS‐YFP‐hα‐Syn as previously reported (Wang et al., 2015 (link)). At 24 h post infection, media were replaced with fresh DMEM and incubated for 48 h. The cell‐free culture media were harvested and centrifuged at 10,000 g using 10 kDa cut‐off filters (Millipore). The resulting supernatants were referred to as α‐Syn‐CM for microglia treatment further. To verify if CM‐contained α‐Syn affected microglial autophagy, CM was pre‐incubated with 2 μg/ml anti‐α‐Syn antibody (#2642, CST) or non‐specific IgG for 10 min before being transferred to microglia culture.
6
Isolation and Characterization of HDL and LDL
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Plasma was collected from consented human participants under active Vanderbilt IRB protocols and guidance. Blood was drawn into EDTA-containing collection tubes and immediately centrifuged to separate plasma. HDL and LDL were isolated from human plasma by KBr density-gradient ultracentrifugation (DGUC), as previously described 32 (link) . Briefly, native LDL (1.019-1.062g/L) and HDL (1.063-1.021g/L) were isolated by sequential DGUC using an Optima XPN-80 Ultracentrifuge with SW41Ti or SW32Ti rotors (Beckman-Coulter). HDL and LDL were dialyzed in PBS with >4 buffer changes and concentrated with 3,000 Da m.w. cutoff filters (Millipore). Total protein levels were determined for each lipoprotein sample (HDL and LDL) by BCA colorimetric assays (Pierce, ThermoFisher).
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