RB1-null C33A and RB1-WT HEK293 cells (107 cells per condition) and SH-SY5Y neuroblastoma cells (207 cells per condition) were cultured in 15-cm dishes, transfected with the indicated expression plasmids, and lysed with ice-cold NP-40 buffer supplemented with protease and phosphatase inhibitors (Roche). All cells were treated with dithiobis succinimidyl propionate (Thermo Fisher Scientific) directly on plastic. Cell lysates were passed 20 times through a 25-gauge needle, incubated on ice for 1 hour, pelleted by centrifugation at 12,000 relative centrifugal force (RCF) for 10 min, precleared by 30 min coincubation with magnetic beads (Life Technologies) at 4°C, and incubated overnight at 4°C with 5 μg of the indicated antibodies per 1 mg of quantified protein lysates. Antibody-protein conjugates were captured by incubation with protein G Dynabeads for 1 hour, washed with ice-cold coimmunoprecipitation buffer, and eluted by boiling in SDS running buffer. Total precleared lysates were used as positive control. Antibodies used for these experiments are listed in data S4.
Dithiobis succinimidyl propionate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dithiobis succinimidyl propionate is a crosslinking agent used in biochemical applications. It is a homobifunctional N-hydroxysuccinimide (NHS) ester that can be used to covalently link primary amine groups on proteins and other molecules.
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Lab products found in correlation
Polyamide substrates, by GE Healthcare (3 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Magnetic bead, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Dylight 594 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Digoxigenin dig, by Roche (1 mentions)
Absolute ethyl alcohol, by Merck Group (1 mentions)
Glucose oxidase antibody, by Abcam (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Sodium l lactate, by Merck Group (1 mentions)
Myriocin, by Merck Group (1 mentions)
Thapsigargin, by Enzo Life Sciences (1 mentions)
Bapta am, by Cayman Chemical (1 mentions)
13 protocols using dithiobis succinimidyl propionate
1
Immunoprecipitation of Protein Complexes
2
Immunoprecipitation and Protein Analysis
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Cells were incubated with 100 µg/ml dithiobis(succinimidyl)propionate (Thermo Fisher Scientific) for 20 min at 37°C and then solubilized in lysis buffer (100-mM Tris-HCl, pH 7.4, 150-mM NaCl, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 2-mM CaCl2, and protease/phosphatase inhibitors) on ice for 30 min. Precleared cell extracts were subjected to antibody precipitation overnight at 4°C, and immune complexes were captured by protein G–Sepharose beads (GE Healthcare). Immunoprecipitated material was separated on Tris-glycine SDS-PAGE, blotted onto nitrocellulose membrane, and analyzed by standard methodologies.
3
Protein Crosslinking and Decrosslinking Protocol
4
Integrating DNA FISH and Histone Modification
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DNA FISH assays were performed as described (Lomvardas et al., 2006 (link)). BAC clones RP24-282B18 and RP11-1122M4, carrying the mouse Jun and human PTGS2 loci, respectively (BACPAC Resources, CHORI, Oakland, CA), were labeled with Digoxigenin (DIG) by nick translation (Roche Diagnostics, Indianapolis, IN) to generate probes for FISH. 10T1/2 cells were grown on coverslips, serum starved for 24 h, and treated with 100 nM TPA for 30 min; CCD-1070Sk cells were serum starved for 48 h and treated with 100 nM TPA for 60 min. After indirect immunolocalization of H3S10ph and H3S28ph, the antigen–antibody complexes were cross-linked with 1 mM dithiobis(succinimidyl propionate) (Thermo Fisher Scientific, Waltham, MA) for 30 min at room temperature. The genomic DNA was denatured using 0.1 N NaOH for 2 min and hybridized to the denatured probes (72°C for 10 min) overnight at 37°C. After extensive washing steps, the probe was detected using primary anti-DIG antibody (Roche Diagnostics) and DyLight 594-conjugated secondary antibody (Jackson ImmunoResearch Laboratories).
5
Fabrication of Enzyme-Functionalized Biosensors
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Polyamide substrates with a pore size of 200 nm and a thickness of 60 µm were obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). The linker molecule dithiobis succinimidyl propionate (DSP), dimethyl sulfoxide (DMSO), and 1X phosphate buffered saline (PBS) were procured from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Salt-free streptavidin from Streptomyces avidiini (≥13 units/mg protein), alcohol oxidase enzyme from Pichia pastoris (10–40 units/mg protein), glucose oxidase from Asperigillus niger (100,000–250,000 units/g), D-(+)-glucose, sodium L-lactate (~98% purity), absolute ethyl alcohol (≥99.5%), and sodium bicarbonate (≥99.7%) were procured from Sigma-Aldrich (St. Louis, MO, USA). NHS-biotin was purchased from Vector laboratories (Burlingame, CA, USA). Glucose oxidase antibody was purchased from Abcam (Cambridge, MA, USA). Lactate oxidase (80 U/mg) was purchased from Toyobo USA. Synthetic sweat was prepared from the recipe described in M.T. Mathew et al. [20 (link)]. The pH range was varied by varying the concentrations of the constituents. Single donor human sweat of pH~6 was purchased from Lee Biosolutions Inc. (Maryland Heights, MO, USA). No preservatives were added to this product and it was stored at −20 °C. All alcohol, glucose, and lactate dilutions were made in synthetic sweat pH 6, 8, and in human sweat buffers.
6
Lipid Reconstitution and Protein Labeling
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N-ethyl maleimide (NEM), dithiothreitol (DTT) and myriocin were from Sigma-Aldrich, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanol-amine (DOPE) and 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoethanolamine (NBD-PE) from Avanti Polar Lipids, and dithiobis(succinimidyl-propionate) (DSP), mouse monoclonal anti-HA agarose beads, C6-NBD-ceramide and N3-Alexa-Fluor647 from Thermo Fischer Scientific. Curcumin and thapsigargin were from Enzo Life Sciences, tamoxifen, fuminosin B1 and BAPTA-AM from Cayman, z-VAD-fmk from Merck Millipore, and L-arabinose from Carl Roth. Mouse monoclonal anti-V5 agarose beads were from Bethyl Laboratories and Ni-NTA agarose from Qiagen.
7
Chloride Sensor Substrate Preparation
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Polyamide substrates (pore size, 200 nm; thickness, 60 µm) were obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). The crosslinking molecule dithiobis succinimidyl propionate (DSP) and its solvent, dimethyl sulfoxide (DMSO), were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The chloride ionophore, 4,5-Bis-[N′-(butyl) thioureido]-2,7-di-tert-butyl-9,9-dimethylxanthene (synonym: Chloride Ionophore IV Selectophore™), was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ionophore solution was prepared by dissolving it in DMSO and vortexing it for 30 min at 1000 rpm. Perspired human sweat of pH 5.98 from a single donor was obtained from Lee Biosolutions Inc. (Maryland Heights, MO, USA) and was stored at −20 °C, without the addition of any preservatives. Potassium chloride, which was used to spike the human sweat samples with the desired chloride levels for sensor calibration, was ordered from Thermo Fisher Scientific Inc. Synthetic sweat (pH 2, 4, 6, and 8) was used as a buffer for zeta potential studies, which was prepared as per the recipe described by Mathew et al., minus the NaCl and KCl dissolution [26 (link)]. Buffer pH was varied using lactic acid (85%) and urea, which were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Thermo Fisher Scientific Inc. (Waltham, MA, USA), respectively.
8
Polyamide-Based Immunosensor Calibration
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Polyamide substrates were ordered from GE Healthcare Life Sciences (Piscataway, NJ, USA) with 0.2 μm pore size. The linker molecule Dithiobis [Succinimidyl Propionate] (DSP) and its solvent Dimethyl Sulfoxide (DMSO) is ordered from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The α-cortisol antibody and cortisol hormone (Hydrocortisone) was ordered from Abcam (Cambridge, MA, USA). IL-1β antigen was bought from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The antibody was diluted in 1X phosphate buffered saline (PBS, Thermo Fisher Scientific Inc., Waltham, MA, USA). Both cortisol hormone and IL-1β are diluted using synthetic sweat. The synthetic sweat was prepared as per the recipe stated in Table 2 of M.T. Mathew et al.26 . The pH range is varied by varying the concentration of the components. Human sweat was purchased from Lee biosolutions Inc. (St. Louis, MO, USA), where it was collected from single human donor with pH ~ 4–5. No preservatives have been added to this product and it was stored unfiltered at below −20 °C.
9
Synthesis and Functionalization of SERS Nanotags
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SERS nanotags were prepared by functionalizing AuNPs with antibodies and Raman reporters and stabilizing with bovine serum albumin (BSA; Life Technologies Australia Pty Ltd.) coatings. Briefly, 60-nm AuNPs were synthesized by citrate reduction of HAuCl4 (39 ). Ten microliters of 1 mM Raman reporters in ethanol (either MBA, TFMBA, DTNB, or MPY) and subsequently 2 μl of 1 mM dithiobis(succinimidyl propionate) (Thermo Fisher Scientific) in dimethyl sulfoxide were added into 1 ml of AuNP solutions and incubated for 5 hours at RT to form a complete self-assembled monolayer. For the functionalization of MPY Raman reporters, 20 μl of 0.1 M NaOH was first added to adjust AuNP solutions to pH = 8. After incubation, the mixture was centrifuged at 7600 rpm for 10 min to remove the residual reactants. The mixture was then resuspended in 200 μl of 0.1 mM PBS and incubated with 2 μg of primary antibodies against either MCSP (R&D Systems, MAB2585), MCAM (R&D Systems, MAB932), ErbB3 (R&D Systems, MAB3481), LNGFR (R&D Systems, MAB367), calnexin (Abcam, ab112995), or CD45 (BioLegend, 368502) for 30 min at RT. The mixture was then centrifuged at 600g at 4°C for 10 min to remove free antibodies and resuspended in 200 μl of 0.1% (w/v) BSA for 0.5 hour at RT to block nonspecific binding sites and stabilize SERS nanotags. The SERS nanotags were stored at 4°C and were stable for months.
10
Biotinylation and Crosslinking of Cell Surface Proteins
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Cells were starved and chilled on ice, rinsed twice with ice-cold biotinylation buffer (PBS, 1 mM CaCl2, 0.5 mM MgCl2), and then incubated (30 min, 4°C) with a membrane-impermeable EZ-Link-Sulfo-NHS-LC-biotin (1 mg/mL; Thermo Scientific, Belgium) to label membrane proteins. Free biotin was quenched (0.1 M glycine, 30 min, 4°C) and washed twice with ice-cold biotinylation buffer. Cells were then incubated in pre-warmed media at 37°C with or without NGF. Cells were then incubated with 1 mM Dithiobis Succinimidyl Propionate (Thermo Scientific) dissolved in hybridization buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.2 mM CaCl2 and 0.2 mM MgCl2) (30 min, 4°C) and then neutralized (20 mM Tris-HCl pH 7.5 and 150 mM NaCl) (30 min, 4°C). This incubation was followed by cell lysis and biotinylated proteins were then isolated by immobilization on streptavidin agarose resins (Thermo Scientific) (3 h, 4°C). Beads were washed three times (lysis buffer, 4°C) and eluted in Laemmli buffer (7 min, 95°C).
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