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Mtset

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MTSET is a chemical reagent that is used for the modification of thiol groups in proteins. It functions by covalently labeling accessible cysteine residues, which can be used to investigate protein structure and function.

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Lab products found in correlation

Mtsea, by Biotium (2 mentions) Sm2 bio beads, by Bio-Rad (2 mentions) 1 2 dioleoyl sn glycero 3 phosphoglycerol, by Avanti Polar Lipids (2 mentions) Hek293t, by American Type Culture Collection (1 mentions) Lipojet, by SignaGen (1 mentions) Hela ccl 2, by American Type Culture Collection (1 mentions) L tryptophan, by Maclin Biochemical Technology (1 mentions) 1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions) Dimethyl sulfoxide (dmso), by Carl Roth (1 mentions) Pitstop 2, by Merck Group (1 mentions) Amiloride hydrochloride, by Merck Group (1 mentions) Carbenoxolone, by Merck Group (1 mentions) Trypsin, by Merck Group (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Aprotinin, by Merck Group (1 mentions) Amiloride, by Merck Group (1 mentions) α chymotrypsin type 2, by Merck Group (1 mentions) Supersignal west femto, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Hela cells ccl 2, by American Type Culture Collection (1 mentions)

9 protocols using mtset

1

Biochemical Characterization of SERT Mutants

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HeLa (CCL-2) and HEK 293T cells were from American Type Culture Collection. C-terminal FLAG-tagged rat SERT C109A, Y107C/C109A, S404C/C109A, and S277C/X5C expression plasmids in pcDNA3.1 were obtained from Dr. Rudnick laboratory, Yale University School of Medicine. Lenti-EF-1α-BSD vector with a blasticidin resistance and two packaging vectors (psPAX2 and pMD2G) were generous gifts from Dr. G. Wang (Guangzhou University). APP+ and ASP+ were purchased from Sigma-Aldrich. Fluoxetine, paroxetine, citalopram, imipramine, 5-HT, 5-hydroxytryptophan (5-HTP) and L-tryptophan were purchased from Macklin. LipoJet was obtained from Signagen. MTSET and MTSEA were purchased from Biotium. All other reagents were of analytical grade.
Li M., Chen Q, & Zhang Y.W. (2022). Determining Ligand and Ion-Induced Conformational Changes in Serotonin Transporter with Its Fluorescent Substrates. International Journal of Molecular Sciences, 23(18), 10919.
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2

Liposome Preparation using DOPC and DOPG

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1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) were obtained from Avanti Polar Lipids Inc. (USA). SM2 Bio-Beads (mesh size 20–50) were purchased from Bio-Rad. MTSET was purchased from Biotium. All other compounds were purchased from Sigma-Aldrich (UK).
Haylock S., Friddin M.S., Hindley J.W., Rodriguez E., Charalambous K., Booth P.J., Barter L.M, & Ces O. (2020). Membrane protein mediated bilayer communication in networks of droplet interface bilayers. Communications Chemistry, 3, 77.
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3

Cldn2 and Cldn10a Labeling in COS-7 Cells

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COS-7 cells were transfected with pSNAP-Cldn2I66C and pYFP-Cldn10a with a total plasmid conc. of 1.6 µg/ml. Cells were either treated with 1 mM MTSET (Biotium, #91021) or the equal volume of DMSO (Roth, #A994.1) for 2 h in serum-free DMEM. Cells were labeled with BG-Atto590 and immunofluorescent stained with α-GFP-NB-Atto647N (1:200) and imaged in two-color STED (see the “Time-gated single- and multi-color STED imaging” section).
Gonschior H., Schmied C., Van der Veen R.E., Eichhorst J., Himmerkus N., Piontek J., Günzel D., Bleich M., Furuse M., Haucke V, & Lehmann M. (2022). Nanoscale segregation of channel and barrier claudins enables paracellular ion flux. Nature Communications, 13, 4985.
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4

Reagents for Membrane Protein Assays

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The sulfhydryl reagent MTSET was obtained from Biotium. Amiloride hydrochloride, connexin inhibitor carbenoxolone, clathrin inhibitor Pitstop-2, and α-chymotrypsin type II from bovine pancreas were purchased from Sigma-Aldrich.
Ilyaskin A.V., Korbmacher C, & Diakov A. (2021). Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis. The Journal of Biological Chemistry, 296, 100404.
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5

MTSET Sensitivity Assay

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The stock solution of MTSET (Biotium) was 20 mM in water and was stored at −20 °C. MTSET at a final concentration of 100 μM was applied either in cis or trans side using pipette manually. The constructs with currents that were not inhibited 5 min after MTSET application were defined as in-sensitive; the constructs whose currents were inhibited and were not restored in 10 min were defined as sensitive.
Xia B., Fang S., Chen X., Hu H., Chen P., Wang H, & Gao Z. (2016). MLKL forms cation channels. Cell Research, 26(5), 517-528.
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6

Cysteine Accessibility Profiling of L5 Strains

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Cultures of the L5::cwlM1cys-strep, L5::lprG-strepC and L5::pgm-strepC strains were grown to late log phase, washed twice and resuspended in PBS + 0.25% tween80, split and treated with 0 or 0.6 mg/ml of MTSEA and MTSET (Biotium, Hayward, CA) for 20 min at RT. Blocking was quenched with cysteine at 50 mM; cells washed in PBS + 0.05% tween80, and resuspended in PEGylation buffer (600 mM Tris pH 7.4, 10 M Urea, 2% SDS, 1 mM EDTA), heated at 85° for 20 min and spun. 5.6 mg of MalPEG5K (Sigma Aldrich) was added to all the supernatants except (-) controls and incubated at 37° for 2 hr. Proteins were TCA precipitated and visualized by α-strep western blot.
Boutte C.C., Baer C.E., Papavinasasundaram K., Liu W., Chase M.R., Meniche X., Fortune S.M., Sassetti C.M., Ioerger T.R, & Rubin E.J. (2016). A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis. eLife, 5, e14590.
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7

Biochemical Reagents for Experiments

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Amiloride, aprotinin, DTT, α-chymotrypsin type II, and trypsin from bovine pancreas were purchased from Sigma–Aldrich. The sulfhydryl reagent MTSET was obtained from Biotium.
Sure F., Bertog M., Afonso S., Diakov A., Rinke R., Madej M.G., Wittmann S., Gramberg T., Korbmacher C, & Ilyaskin A.V. (2022). Transmembrane serine protease 2 (TMPRSS2) proteolytically activates the epithelial sodium channel (ENaC) by cleaving the channel’s γ-subunit. The Journal of Biological Chemistry, 298(6), 102004.
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8

Albizia Julibrissin Bark Extract Evaluation

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The bark of Albizia Julibrissin Durazz. was obtained from Anhui Xiehecheng Pharmaceutical Co. Ltd., Anhui, China, (batch no. 19120704). HeLa cells (CCL-2) were from American Type Culture Collection. N-terminal His6 and C-terminal FLAG-tagged human SERT (hSERT), C109A, and Y107C/C109A expression constructs used in this study were described previously [37 (link),67 (link)]. Expression plasmids for human DAT (hDAT) and human NET (hNET) in pcDNA3.1 were from the Dr. Rudnick lab (Yale School of Medicine). APP+, ASP+, protease inhibitor mixture cocktail, anti-Flag monoclonal M2 antibody, fluoxetine, GBR 12909, and desipramine were purchased from Sigma-Aldrich. EZ-Link™ NHS-SS-Biotin, streptavidin-agarose, Super Signal West Femto, lipofectamine 2000, and Micro BCA protein assay reagent kit were obtained from Thermo Fisher Scientific. MTSET was purchased from Biotium. Cell counting kit-8 (CCK-8) was from Dojindo (Shanghai, China). All other reagents were of analytical grade.
Huang B., Liu H., Wu Y., Li C., Tang Q, & Zhang Y.W. (2022). Two Lignan Glycosides from Albizia julibrissin Durazz. Noncompetitively Inhibit Serotonin Transporter. Pharmaceuticals, 15(3), 344.
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9

Reconstitution of Lipid Membranes

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1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) were obtained from Avanti Polar Lipids Inc. (USA). SM2 Bio-Beads (mesh size 20-50) were purchased from BioRad. MTSET was purchased from Biotium. All other compounds were purchased from Sigma-Aldrich (UK).
Haylock S., Friddin M.S., Hindley J.W., Rodriguez E., Charalambous K., Booth P.J., Barter L.M, & Ces O. (2020). Membrane protein mediated bilayer communication in networks of droplet interface bilayers. Communications Chemistry, 3, 77.
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