Cell pellets were lysed in 50 mM Tris (pH 8.0), 200 mM NaCl, 4 mM MgCl2, 0.5% Tween-20, 5 mM DTT, 1 mM ATP, 0.2 mM PMSF, and 1× protease inhibitor cocktail (catalog no.: 11873580001) and mechanically dounced preceding a 25 min centrifugation at 39,000g. The supernatant was collected and filtered through 5 and 1.2 μM filters. The filtered supernatant was applied to a column containing SulfoLink resin (Thermo Fisher; catalog no.: 20402) coupled to PDZ. The flow through was collected, and the column was washed with 30 mM Tris (pH 7.5), 50 mM KCl, 5 mM MgCl2, 1 mM DTT, and 1 mM ATP. Myosin S1 was eluted using a peptide with higher PDZ specificity (Genescript, Trp-Glu-Thr-Trp-Val). Recombinant myosin-S1 was dialyzed against storage buffer containing 20 mM Mops (pH 7.0), 25 mM KCl, 5 mM MgCl2, and 10% sucrose. Proteins were flash frozen after the addition of 1 mM DTT and 1 mM ATP and stored at −80 °C. The resulting purified protein consists of recombinant myosin-S1 with endogenous mouse C2C12 light chains.
Sulfolink resin
Manufactured by Thermo Fisher Scientific
Sourced in France
SulfoLink resin is a solid-phase matrix designed for the immobilization of proteins, peptides, or other molecules containing free sulfhydryl groups. The resin provides a covalent coupling method for attaching these molecules to a solid support.
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Lab products found in correlation
Sulfolink coupling resin, by Thermo Fisher Scientific (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 protein labeling kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 protein labeling kit, by Thermo Fisher Scientific (1 mentions)
Sepharose 4b, by Merck Group (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Pd 10 column, by Cytiva (1 mentions)
Protease phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Np 40, by Thermo Fisher Scientific (1 mentions)
Fm4 64, by Thermo Fisher Scientific (1 mentions)
Texas red labeled antimouse, by Vector Laboratories (1 mentions)
Tetramethyl rhodamine isothiocyanate tritc labeled phalloidin, by Merck Group (1 mentions)
Ab117819, by Abcam (1 mentions)
Ma5 15738, by Thermo Fisher Scientific (1 mentions)
Sc 23954, by Santa Cruz Biotechnology (1 mentions)
Ma5 15256, by Thermo Fisher Scientific (1 mentions)
Miniprep kit, by Qiagen (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Sanger sequencing, by Genewiz (1 mentions)
Hsp90, by Santa Cruz Biotechnology (1 mentions)
21 protocols using sulfolink resin
1
Recombinant Myosin-S1 Purification
2
Production and Purification of Anti-SOD1 Antibodies
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Production of a polyclonal antibody to a peptide of SOD1 (Gly 44 – Asn 53) was performed by Eurofins Genomics. Briefly, the peptide, H2N-CG44FHVHEFGDN53-COOH, was conjugated through its N-terminal Cys with keyhole limpet hemocyanin, with which a rabbit was immunized in the 42-day protocol. The sera were then purified using a Sulfo-Link Coupling Resin (Thermo) with the peptide, Gly 44 – Asn 53, Gly 44 – Glu 49, His 46 – Gly 51, or His 48 – Asn 53, by which anti-SOD144–53, anti-SOD144–49, anti-SOD146–51, or anti-SOD148–53 antibody was purified, respectively. All of the peptides have an additional Cys residue at the N-terminus for its conjugation with the resin. For preparation of anti-SOD1int antibody, anti-SOD144–53 antibody was first loaded on a Sulfo-Link Coupling Resin (Thermo) cross-linked with purified apo-SOD1S-S proteins, and the flow-through fraction was collected, concentrated, and then purified using a Sulfo-Link resin conjugated with a His 48 – Asn 53 peptide. Concentrations of purified antibodies were determined by Micro BCA Protein Assay kit (Thermo).
3
Phosphoantibody Generation and Labeling
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For the generation of rabbit polyclonal phosphoantibodies, synthetic phosphopeptides were used for immunization (Young In Frontier, South Korea). Phosphoantibodies were affinity-purified using a corresponding phospho-peptide immobilized to SulfoLink resin (Thermo Fisher Scientific). Alexa Fluor 594-conjugated anti-Plk4 pSSTT antibody was generated using the Alexa Fluor 594 protein labeling kit (Molecular Probes). The list of phosphopeptides and their respective nonphospho-peptides used in this study is provided in Supplementary Table 4 .
An anti-Cep152 (491–810) antibody4 (link) was conjugated with Alexa Fluor 647 by using the Alexa Fluor 647 protein labeling kit (Molecular Probes).
An anti-Cep152 (491–810) antibody4 (link) was conjugated with Alexa Fluor 647 by using the Alexa Fluor 647 protein labeling kit (Molecular Probes).
4
Antibody Production Against XMAP4 Domains
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For production of antibodies against the MBD of XMAP4, a fragment of DNA that encoded amino acids 722–1228 of XMAP4 was amplified by PCR using GGAATTCCATATGGCA GAACCT GCTGCTGCAGC as forward primer and CCGCTCGAGTTA GATGCTTGT CTCTGG TATTAG as reverse primer. The PCR product was cloned into the bacterial expression vector pCold I DNA (Clontech) using the Nde and XhoI restriction sites. Recombinant protein was expressed in Escherichia coli according to instructions provided by the vector manufacturer, purified by affinity chromatography on Ni-NTA agarose (Qiagen), dialyzed against PBS, and used for rabbit immunization. High-titer antisera were produced by Bio-Synthesis (Lewisville, TX). Polyclonal antibodies were purified from antisera by affinity chromatography on a CNBr-activated Sepharose 4B (Sigma-Aldrich, St. Louis, MO) with covalently attached antigen. Purified antibodies were dialyzed against microinjection buffer and concentrated by ultrafiltration.
Polyclonal antibodies against XMAP4 projection domain were generated by immunization of rabbits with the synthetic peptide GCDDDDVKEPKNKSERSAAPHD manufactured by Bio-Synthesis, which corresponded to the amino acid residues 56–75 of XMAP4. Antibodies were purified by affinity chromatography of antisera on SulfoLink resin (Thermo Scientific, Rockford, IL) with covalently attached peptide antigen.
Polyclonal antibodies against XMAP4 projection domain were generated by immunization of rabbits with the synthetic peptide GCDDDDVKEPKNKSERSAAPHD manufactured by Bio-Synthesis, which corresponded to the amino acid residues 56–75 of XMAP4. Antibodies were purified by affinity chromatography of antisera on SulfoLink resin (Thermo Scientific, Rockford, IL) with covalently attached peptide antigen.
5
Recombinant Myosin Production Protocol
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Recombinant myosin was produced as described previously with minor changes (22 , 27 (link), 63 (link), 64 (link)). Briefly, adenoviruses encoding myosin S1 were used to infect C2C12 cells 3 days after differentiation. Cells were collected 4 days after infection, pelleted, collected in liquid nitrogen, and stored at −80 °C. Cells were thawed and lysed in 50 mM Tris (pH 8.0), 200 mM NaCl, 4 mM MgCl2, 0.5% Tween-20, 5 mM DTT, 1 mM ATP, 0.2 mM PMSF, and 1× protease inhibitor cocktail (MilliporeSigma/Roche catalog no.: 11873580001), dounce homogenized, and then spun down at 39,000g. The supernatant was filtered through 5 and 1.2 μM filters and applied to a SulfoLink resin (ThermoFisher; catalog no.: 20402) coupled to PDZ. The column was washed with a buffer containing 30 mM Tris (pH 7.5), 50 mM KCl, 5 mM MgCl2, 1 mM DTT, and 1 mM ATP. The WETWV (Genescript) peptide was used to elute myosin S1, which was dialyzed against a storage buffer containing 20 mM Mops (pH 7.0), 25 mM KCl, 5 mM MgCl2, and 10% sucrose. 1 mM DTT and 1 mM ATP were added to the proteins, and they were frozen in liquid nitrogen and stored at −80 °C.
6
Polyclonal Antibody Purification Protocol
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Tagless SOA(1–122) was re-buffered in PBS using a PD-10 column (Cytiva) for immunization. Immunization was carried out by Eurogentec using their polyclonal 28-day speedy programme. For epitope purification of the SOA antibody from the serum, 2 ml sulfolink resin (Thermo Fisher Scientific) was covalently conjugated with 3 mg tagless SOA(1–122) according to the manufacturer’s protocol. Next, 10 ml final bleed was incubated with the SOA(1–122)-conjugated sulfolink resin at 4 °C overnight while rotating. After incubation, the resin was washed with PBS containing 0.1% Triton X-100, followed by PBS in a gravity-flow poly-prep column (Bio-Rad). Elution was performed using low pH (100 mM glycine-Cl and 150 mM NaCl, pH 2.3) followed by immediate neutralization of elution fractions with Tris-Cl pH 8.0. The eluted antibody was re-buffered using a PD-10 column (PBS, 0.05% NaN3 and 10% glycerol) and concentrated to 1 mg ml–1 using an Amicon spin-concentrator before flash-freezing in liquid nitrogen and storage at −80 °C.
7
Affinity Purification of Phospho-ITIM Interactors
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Peptides corresponding to the tyrosine-phosphorylated ITIM of Ly49Q (CGGMSEQEVTpYSTVRFHK) and to the nonphosphorylated ITIM (CGGMSEQEVTYSTVRFHK) were synthesized by Operon Biotechnologies Inc.) and conjugated to SulfoLink resin according to the manufacturer’s instructions (Thermo Fisher Scientific). Lysates of Raw264.7 cells were prepared with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% glycerol, 150 mM NaCl, 1% NP-40, and protease/phosphatase inhibitors (Thermo Fisher Scientific) and incubated with the peptide-conjugate resins for 1 h at 4°C. The resins were then washed with lysis buffer, and the proteins bound to the resin were separated by SDS−PAGE and visualized by silver staining. Proteins specifically bound to the phosphorylated ITIM peptides were excised and subjected to liquid chromatography-tandem mass spectrometry (LC−MS/MS; APRO Science Inc.).
8
Recombinant Mouse PIERCE1 Protein Purification
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Recombinant 6xHis-tagged mouse PIERCE1 protein was expressed in BL21(DE3)pLysS competent cells (Novagen) and purified by Ni-NTA affinity chromatography. Purified protein was injected into New Zealand White rabbits (by Covalab, France); antibodies were purified from rabbit sera by affinity purification using PIERCE1 recombinant protein bound to SulfoLink resin (Thermo Fisher Scientific, Cat #20401).
9
Antibody Purification and Fluorescent Labeling
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The anti-FMNL3 antibody was raised in guinea pig (Covance) using the FMNL3-FH1FH2 construct (Harris et al., 2010 (link)) as antigen and affinity-purified using this same antigen coupled to sulfolink resin (Thermo Scientific, Waltham, MA). Anti–N-cadherin (mouse, #C3845) and anti-tubulin (mouse, DM1alpha) antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Alexa 488 and Alexa 546–labeled anti–guinea pig secondary antibody and FM4-64 were purchased from Life Technologies. Texas Red–labeled anti-mouse was obtained from Vector Laboratories. Tetramethylrhodamine isothiocyanate (TRITC)–labeled phalloidin was purchased from Sigma-Aldrich. Alexa 647–WGA (Life Technologies) was a kind gift from Brent Berwin (Geisel School of Medicine at Dartmouth).
10
Antibody Production for Phospho-aPKCι Analysis
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The phospho-Tyr-136 antibody used in this study was made using an immunogenic peptide spanning residues 125 to 141 of human aPKCι synthesized with modifications at Tyr-136 (phosphate) and Cys-137 (S-methylcarboxyamide) and included an additional N-terminal Cys for coupling reactions. New Zealand White rabbits were injected (off-site on commission by White Antibodies) with KLH coupled antigen (500 μg) and Freund’s complete adjuvant and subsequently on day 14, 28, 42, 56, and 70 with antigen and Freund’s incomplete adjuvant. The serum was collected on day 77, and reactive antibodies were purified from serum by sequential purification on a nonphosphopeptide column (flowthrough) and a phospho-peptide column (eluate). These were prepared by binding peptides to SulfoLink resin according to the protocol of the manufacturer (Thermo Fisher Scientific). Antibodies were eluted from the phospho-peptide column using 100 mM glycine pH 3.0 and immediately neutralized with 1 M Tris-HCl, pH 8.
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