In vitro binding experiments were performed with recombinant GST-TMIGD1 fusion proteins purified from E.coli and immobilized on glutathione-Sepharose 4B beads (Life Technologies #17-0756-01). Purification of GST fusion proteins was performed as described80 (link). For the analysis of direct protein interactions, Scrib constructs were translated in vitro using the TNT T7-coupled reticulocyte lysate system (Promega Corp., Madison, WI) in the presence of 35[S]-labeled methionine (Hartmann Analytic GmbH, Braunschweig, Germany) as described by the manufacturer. 10 µl of the translation reactions were incubated with 3 µg of immobilized GST fusion protein for 2 h at 4 °C under constant agitation in buffer B (10 mM Hepes-NaOH (pH7.4), 100 mM KCl, 1 mM MgCl2, 0.1% Triton X-100). After 5 washing steps in buffer B bound proteins were eluted by boiling for 5 min in SDS sample buffer, subjected to SDS-PAGE and analyzed by fluorography. All in vitro binding experiments shown in this study are representative for at least three independent experiments.
Glutathione sepharose 4b beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Glutathione Sepharose 4B beads are a chromatography resin composed of cross-linked agarose beads with immobilized glutathione. The beads are designed for the purification of glutathione S-transferase (GST)-tagged recombinant proteins from crude cell lysates or other biological samples.
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Lab products found in correlation
Tnt t7 coupled reticulocyte lysate system, by Promega (3 mentions)
35s labeled methionine, by Hartmann Analytic (1 mentions)
Escherichia coli bl21, by GE Healthcare (1 mentions)
Streptavidin beads, by Merck Group (1 mentions)
Luria bertani medium, by Merck Group (1 mentions)
Lysozyme, by Yeasen (1 mentions)
Ultrasonic crusher, by Ningbo Scientz Biotechnology (1 mentions)
Laemmli loading buffer, by Bio-Rad (1 mentions)
Rhotekin rbd, by Cytoskeleton (1 mentions)
Millipore concentrator, by Merck Group (1 mentions)
Nh4cl, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions)
Reduced l glutathione, by Thermo Fisher Scientific (1 mentions)
Thrombin protease, by MP Biomedicals (1 mentions)
Ni nta beads, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d thiogalactoside, by Merck Group (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Nickel nitrilotriacetic acid ni nta beads, by Thermo Fisher Scientific (1 mentions)
Proteinase inhibitor cocktail tablet, by Roche (1 mentions)
13 protocols using glutathione sepharose 4b beads
1
In Vitro Protein Interaction Analysis
2
GST Pull-down Assay for Protein Interactions
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In vitro binding experiments were performed as described previously (Ebnet et al., 2000 (link)). Briefly, GST-fusion proteins were expressed in Escherichia coli BL21 (GE Healthcare). Bacteria were lysed by passaging through a French pressure cell, and GST-fusion proteins were purified by affinity chromatography. Protein solutions were adjusted to 50% (wt/vol) glycerol and stored at −20°C. For GST pull-down experiments, the prey proteins were generated in vitro using the TNT T7-coupled reticulocyte lysate system (Promega, Madison, WI) in the presence of 35[S]methionine as described by the manufacturer. We incubated 10 μl of the translation reactions with 3 mg of GST-fusion proteins immobilized on glutathione-Sepharose 4B beads (Life Technologies) for 2 h at 4°C under constant agitation. Beads were washed five times with buffer B (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.2, 100 mM KCl, 1 mM MgCl2, 0.1% Triton X-100). Bound proteins were eluted by boiling for 5 min in SDS sample buffer, subjected to SDS–PAGE, and analyzed by fluorography.
3
In vitro Protein-Protein Interaction Assay
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In vitro binding experiments were performed with either recombinant GST-fusion proteins purified from E. coli and immobilized on glutathione-Sepharose 4B beads (Life Technologies) or with biotinylated peptides immobilized on streptavidin beads (Sigma-Aldrich). Purification of GST fusion proteins was performed as described (Ebnet et al., 2000 (link)). For in vitro interactions the putative partner proteins (prey) were translated in vitro using the TNT T7-coupled reticulocyte lysate system (Promega Corp.) in the presence of 35[S]-labeled methionine, as described by the manufacturer. Ten microliters of the translation reactions were incubated with 3 µg of immobilized GST fusion protein or with 0.5 µg of peptide immobilized on streptavidin beads for 2 h at 4°C under constant agitation in buffer B. After five washing steps in buffer B, bound proteins were eluted by boiling for 5 min in SDS sample buffer, subjected to SDS-PAGE, and analyzed by fluorography.
4
GST-Fusion Protein Binding Assay
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In vitro binding experiments were performed with recombinant GST-fusion proteins purified from E.coli and immobilized on glutathione-Sepharose 4B beads (Life Technologies). Purification of GST fusion proteins was performed as described [42 (link)]. For protein interaction experiments the putative partner protein (prey) was expressed in HEK293T cells by transient transfection. Cells were lysed as described for immunoprecipitations. Lysates were incubated with 3 μg of immobilized GST fusion protein for 2 h at 4 °C under constant agitation. After 5 washing steps in lysis buffer, bound proteins were eluted by boiling for 5 min in SDS sample buffer, subjected to SDS-PAGE and analyzed by Western blotting using prey-specific antibodies.
5
GST Fusion Protein Pull-Down Assay
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In vitro binding experiments were performed with recombinant GST fusion proteins purified from E.coli and immobilized on glutathione-Sepharose 4B beads (Life Technologies #17-0756-01). Purification of GST fusion proteins was performed as described (Ebnet et al., 2000) . For protein interaction experiments the putative partner protein (prey) was expressed in HEK293T cells by transient transfection. Cells were lysed as described for immunoprecipitations. Lysates were incubated with 3 µg of immobilized GST fusion protein for 2 h at 4°C under constant agitation. After 5 washing steps in lysis buffer, bound proteins were eluted by boiling for 5 min preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted February 4, 2021. ; https://doi.org/10.1101/2021.01.25.428038 doi: bioRxiv preprint in SDS sample buffer, subjected to SDS-PAGE and analyzed by western blotting using preyspecific antibodies.
The copyright holder for this this version posted February 4, 2021. ; https://doi.org/10.1101/2021.01.25.428038 doi: bioRxiv preprint in SDS sample buffer, subjected to SDS-PAGE and analyzed by western blotting using preyspecific antibodies.
6
Rac1 Activity Measurement in VSMCs
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Rac1 activity was measured by GTP pull‐down assay. Briefly, VSMC were lysed with buffer containing 50 mmol/L of Tris‐HCl (pH 7.40, 1 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulphonyl fluoride, 1% Triton X‐100, 1 mM sodium fluoride and proteinase inhibitor cocktail tablet). Lysates were incubated with glutathione S‐transferase/p21‐activated protein kinase/Rac interactive binding domain (GST‐PAK‐CRIB) and glutathione Sepharose‐4B beads (Thermo Fisher Scientific). Beads were then briefly washed. Bound proteins were eluted by boiling in 2× SDS sample buffer and then subjected to 12% SDS‐PAGE followed by immunoblotting with the Rac1 antibody. Blots were analysed with densitometry.
7
Fluorescent DNA Binding Assay
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GST-tagged MORFWH2 (100 μM) was incubated with glutathione Sepharose 4B beads (Thermo Fisher Sci) at 4 °C for 0.5–1 h then washed with buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5 mM BME). Buffer was removed and the beads were resuspended in 1:1 washing buffer. To prepare for imaging, 10 μM fluorescein (FAM)-labeled 37 bp dsDNA (10–20 μM) was incubated with 10 μM of the suspended beads for 0.5–1 hour at room temperature. Confocal images were acquired on a Zeiss Observer.Z1 inverted microscope using a 488 nm laser for the excitation and emission of FAM. Images were processed using ImageJ.
8
PD-L1 Protein Expression and Purification
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Glutathione S‐transferase (GST)‐tagged PD‐L1 and its corresponding truncated mutants (GST‐PD‐L1‐E, GST‐PD‐L1‐IgV, GST‐PD‐L1‐IgC2 and GST‐PD‐L1‐C) (Sangon Biotech) underwent cloning into the pGEX‐6p‐1 vector (Biosettia) prior to expression in E. coli BL21 cells (WeiDi, Shanghai, China). Cell induction employed 0.5 mmol/L isopropyl β‐D‐thiogalactoside (IPTG) (Yeasen) in Luria‐Bertani (LB) medium (Sigma, St. Louis, MO, USA) at 16°C for 24 h to induce PD‐L1 protein expression. The cell pellet was dissolved in lysis buffer (50 mmol/L Tris, 300 mmol/L NaCl), and 2 mg/mL lysozyme (Yeasen) was added and incubated at 4°C overnight, followed by ultrasonication using Ultrasonic Crusher (SCIENTZ, Ningbo, Zhejiang, China), and a 20 min centrifugation at 18,000 × g at 4°C. Following removal of supernatant, the inclusions were collected in solubilization buffer [200 mmol/L Tris, 5 mmol/L dithiothreitol (DTT), 8 mol/L urea, pH = 8.0]. Insoluble material was centrifuged at 18,000 × g for 20 min at 4°C and removed, and the supernatant was placed onto Glutathione Sepharose 4B beads (ThermoFisher Scientific) and equilibrated with binding buffer (50 mmol/L Tris, 300 mmol/L NaCl) overnight. The protein was eluted with elution buffer (50 mmol/L Tris, 300 mmol/L NaCl, 10 mmol/L glutathione, pH = 7.5‐8.0) and stored at ‐80°C.
9
Quantifying RhoA and Rac1 Activation
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Rhotekin-RBD (rho binding domain) beads (Cytoskeleton, Denver, CO, USA) were used to precipitate activated GTP-bound RhoA from retinal samples. To precipitate active GTP-bound RAC1, the p21 binding domain of Pak3 (PAK-GST-PBD)25 (link) expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and immobilized by binding to glutathione-Sepharose 4B beads (Thermo Scientific) was used. Retinal samples were lysed as described above (in Western Blotting) and immediately added to 40 μg of Rhotekin-RBD beads or 60 μg of PAK-GST-PBD beads. The mixture was incubated for 1 hour at 4°C, then centrifuged for 2 minutes at 5000 rpm at 4°C, washed once with excess lysis buffer and twice with phosphate buffer saline (PBS), resuspended in 40 μL of 2× Laemmli loading buffer (Bio-Rad), boiled for 3 minutes, and stored at −20°C. RhoA and RAC1 activation assay samples and total retinal protein were analyzed by standard Western blot techniques.
10
Mutational Analysis of MORFDFP Protein
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MORFDPF mutants (F287A, D289A, F218A, R276E, R306E/K309E, and F287A/R306E/K309E) were generated using the Quick Change site-directed lightning mutagenesis protocol (Stratagene) (Supplementary Table 2 ). Unlabeled and 15N-labeled WT and mutated MORFDPF (residues 211–322) were expressed in E. coli Rosetta2 (DE3) pLysS cells grown in LB or NH4Cl (Sigma-Aldrich) minimal media supplemented with 50 µM ZnCl2. After induction with IPTG (final concentration 0.5 mM) (Gold Biotechnology) for 16 h at 18 °C, cells were harvested via centrifugation and lysed by freeze-thaw followed by sonication. The unlabeled and 15N-labeled GST-fusion proteins were purified on glutathione Sepharose 4B beads (Thermo Fisher Science). The GST tag was either cleaved with Thrombin protease (MP Biomedicals), or left on and the proteins were eluted off the resin with 50 mM reduced l -glutathione (Fisher). The proteins were concentrated into PBS buffer pH 6.5, supplemented with 5 mM dithiothreitol (DTT). The unlabeled proteins were purified by size exclusion chromatography and concentrated in Millipore concentrators (Millipore).
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