Samples were homogenized in immunoprecipitation buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, and 1 mM DTT) containing 1 mM PMSF and a protease inhibitor mixture. The homogenized samples were sonicated using a Branson 250 Digital Sonifier with 1-second-on and 1-second-off pulses at 40% power amplitude for 15 seconds. Precleared lysates were incubated with the relevant primary antibody–conjugated magnetic beads for 16 hours at 4°C. Immune complexes were collected, washed 4 times with immunoprecipitation buffer, and applied to 4%–12% SDS-polyacrylamide gels for Western blot analysis before transferring to PVDF membranes. Primary antibodies against HDAC3 (1:1,000), Tal1 (1:1,000), Gata2 (1:1,000), and Ets1/2 (1:1,000) were used and visualized by chemiluminescence using HRP-conjugated secondary antibodies. Blots were probed with α-tubulin (1:1,000) for the loading control.
Digital sonifier 250
Manufactured by Emerson
Sourced in United States
The Digital Sonifier 250 is a laboratory instrument designed for sample preparation and homogenization. It utilizes ultrasonic energy to disrupt cells, disperse particles, and mix solutions. The device features digital controls for precise adjustment of power and pulse settings, enabling users to optimize the process for their specific application.
Automatically generated - may contain errors
Lab products found in correlation
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Proteasemax surfactant, by Promega (1 mentions)
Hypotonic lysis buffer, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Trichloroacetic acid tca, by Merck Group (1 mentions)
Flag m2 antibody, by Merck Group (1 mentions)
Ez chip assay kit, by Merck Group (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose 4b resin, by GE Healthcare (1 mentions)
L glutathione reduced, by Merck Group (1 mentions)
Pure ethanol, by ITW Reagents (1 mentions)
Trans blot turbo mini pvdf transfer pack, by Bio-Rad (1 mentions)
Mini protean precast gel, by Bio-Rad (1 mentions)
Supersignal chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Sf 900 2 sfm, by Thermo Fisher Scientific (1 mentions)
Anhydrous pyridine, by Merck Group (1 mentions)
Thermo 1300 gc, by Thermo Fisher Scientific (1 mentions)
34 protocols using digital sonifier 250
1
Immunoprecipitation and Western Blot Analysis
2
Insect Tissue Protein Extraction and Digestion
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Gut tissue from adult psyllids was divided into replicate samples of 250 each and collected using centrifugation and re-suspended in 10% TCA in acetone for a protein extraction method our lab has optimized for insect tissues [37 (link)] with the following modifications. Tissue was disrupted for two rounds of 10 sec each using a Branson 250 digital sonifier at 10% total amplitude. Total protein was precipitated at -20°C overnight. The precipitated proteins were reconstituted with proteaseMAX surfactant (Promega, Madison, WI). Protein concentration was quantified using a Bradford Assay and confirmed using densitometry on 10% polyacrylamide gels by comparing to a BSA loading control. Proteins were reduced with 5mM tris(2-carboxyethyl)phosphine at 55°C for 20 min, then alkylated with 33mM methyl methanethiosulfonate at room temperature for 20 min. Each sample containing 200μg of protein was digested using 4μg of trypsin at 37°C overnight. The protein digests were dried using a vacuum concentrator prior to mass spectrometry analysis.
3
Preparation of Cotton and Wood CNCs
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Native CNCs from cotton (cCNCs). Cotton linters were treated with 65 wt. % sulfuric acid during 30 min at 63 °C under mechanical stirring following the protocol described by Revol et al. (Revol et al., 1992) . The resulting suspension was washed by repeated centrifugation/redispersion cycles and dialyzed against distilled water until the conductivity of the dialysis bath reached the conductivity of distilled water. In the following step, the suspension was ultrasonicated for 4 min with a Branson 250 digital sonifier and successively filtered through 8 µm and 1 µm cellulose nitrate membranes using a Sartorius filtration equipment. These native cellulose CNCs obtained from cotton will be referred to as cCNCs.
Native CNCs from wood (wCNCs). A 3 wt. % aqueous dispersion of neutral CNCs was provided by Melodea Ltd. (Israel) and used after dilution to 1 wt. % with deionized water. These nanocrystals, that will be referred to as wCNCs, were produced from wood by the reported sulfuric acid (64 %) hydrolysis method (Bondeson, Mathew, & Oksman, 2006) .
Native CNCs from wood (wCNCs). A 3 wt. % aqueous dispersion of neutral CNCs was provided by Melodea Ltd. (Israel) and used after dilution to 1 wt. % with deionized water. These nanocrystals, that will be referred to as wCNCs, were produced from wood by the reported sulfuric acid (64 %) hydrolysis method (Bondeson, Mathew, & Oksman, 2006) .
4
Thioureation of Cellulose Nanocrystals
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Thioureation refers to the reaction that introduces the thiourea group. An aqueous solution containing 10 wt. % HAc and 2 wt. % thiosemicarbazide was prepared. Then, a suspension containing about 2 g of cellulose was mixed with the previous solution in the same volume in a flask surmounted by a condenser. The mixture was heated at 60 °C for 3 h and finally cooled in ice. Then, NaCl was added until its concentration was approximately 0.4 M. The suspension was then centrifuged for 30 min at 11200 rpm and the modified CNCs were redispersed in HAc. The latter operation was repeated twice.
The final product, hereafter referred to as S-e-CNC, was dialyzed against distilled water until constant conductivity of the dialysis bath and ultrasonicated for 4 min with a Branson 250 digital sonifier.
The final product, hereafter referred to as S-e-CNC, was dialyzed against distilled water until constant conductivity of the dialysis bath and ultrasonicated for 4 min with a Branson 250 digital sonifier.
5
Histone Isolation and Purification from Oli-Neu Cells
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The histone component of Oli-Neu nuclei was isolated and purified by using the acid extraction protocol (Shechter et al., 2007 (link)). The 5 × 106 cells were lysed in 1 ml of hypotonic lysis buffer (10 mM of Tris-Cl pH 8.0, 1 mM of KCl, 1.5 mM of MgCl2, 1 mM of DTT, and 1X protease and phosphatase inhibitor cocktails; all from Sigma-Aldrich, St Louis, MO, United States). After 30 min at 4°C in mild shaking to favor hypotonic swelling and lysis, intact nuclei were pelleted 10,000 × g for 10 min at 4°C, resuspended in 400 μL of 0. N H2SO4, and incubated for 30 min in rotation. After centrifugation at 16,000 × g for10 min at 4°C, 100% of trichloroacetic acid (TCA; Sigma-Aldrich, St Louis, MO, United States) was added dropwise to the supernatant to allow histones precipitation overnight at 4°C. The following day, the solution was centrifuged at 16,000 × g for 10 min at 4°C and the pellet was washed twice in glacial acetone, dried at room temperature, and resuspended in phosphate-buffered saline (PBS) with 1X protease and phosphatase inhibitor cocktails. All samples were sonicated with a Branson 250 digital sonifier, before quantification for subsequent Western Blot (WB) analysis.
6
Preparation of Cellulose Nanocrystal Suspensions
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CNC-II suspensions were prepared according to the method described by Flauzino Neto et al. 12 Cotton linters were treated with a 20 wt % sodium hydroxide aqueous solution under mechanical stirring for 5 h at 25 °C, using 30 mL of solution per gram of material. The material was washed several times with distilled water until the alkali was completely removed, and dried at 40 °C for 12 h in an aircirculating oven. The mercerized cotton linter was milled and submitted to sulfuric acid hydrolysis. Here, for each gram of mercerized cotton linter, 20 mL of a 60 wt % H2SO4 were used. The hydrolysis was performed at 45 °C for 50 min under vigorous stirring. The suspensions were washed by repeated centrifugations, dialyzed against distilled water until constant conductivity of the dialysis bath. The suspensions were then ultrasonicated five times 2 min with a Branson 250 digital sonifier. The resulting suspension had a concentration of 2.4 wt %. Suspensions of higher concentration for rheological experiments were prepared as follows. The pH of a 1 wt % CNC-II suspension containing 3 g of dry CNCs was adjusted to 7 by adding an 0.1 M NaOH solution. The suspension was then freeze-dried and the CNC-II were redispersed in distilled water to prepare aqueous CNC-II samples at a given concentration in the 1-5 wt % range. Sample homogeneity was ensured by ultrasonicating three times for 2 min.
7
Isotopic Labeling of Human USP14 and Domains
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Non‐isotopically 6xHis‐tagged human USP14 and its two domains were produced as previously described (Selvaraju et al., 2019 (link)). 13C‐ and 15N‐labeled USP141–80 and USP141–494 was cloned into pNIC‐28‐Bsa4 vector and expressed in E. coli BL21(DE3) Ros2 cells and grown in M9 minimal medium enriched with 13C‐D‐glucose and 15NH4Cl, at 37°C until OD600 = 0.6. The culture was induced with 0.5 mM IPTG and harvested after 18 h at 18°C. The pellet was resuspended in 20 mM HEPES pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% glycerol, 0.5 mM TCEP, 5 units/mL recombinant DNAse I and 1 EDTA‐free protease inhibitor cocktail tablet per 75 mL, and lysed by sonication using a Branson Digital Sonifier 250 for 3 min with the parameters 10 s on, 10 s off, and 30% amplitude. The purification followed the same procedure as non‐isotopically labeled USP14.
8
Curcumin Sonication with ASL
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CU (figure 1 a) was purchased from Sigma Aldrich (approx. 99% purity). Curcumin (1 mg ml−1) was probe-sonicated for 40 min using Branson Digital Sonifier 250 together with different concentrations of ASL from 1 to 5 w/v% (above CMC = 0.11 mg ml−1)[56 (link)].
9
Tissue Metabolite Extraction and Purification
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Tissue pieces were disrupted in ice-cold 60% MeOH (typically 500 μL per 10 mg tissue) using microtube pestle homogenizer (10s) (Nippon Genetics, cat. no. NG010) or 20 strokes with roughened glass-to-glass potter homogenizer (for heart, skeletal muscle, and kidney) followed by sonication (12s, amplitude 10%, Branson Digital Sonifier 250) to complete the homogenization. Cell pellets were directly sonicated. Further precipitation of proteins was achieved by addition of 225 μL chloroform per 500 μL homogenate and centrifugation (3–6 min 18000 g at 4°C). The aqueous phase was collected and washed three times with ∼1.4 mL diethyl ether to remove MeOH. Any remaining layer of diethyl was evaporated by gentle ∼10s flow of N2 gas. The remaining traces of diethyl ether were evaporated at 65°C in centrifugal vacuum evaporator (SpeedVac Plus SC110A, Savant Instruments) for 5–6 min. The extract volume was estimated by weighing the extract and assuming density of 1 mg/μL. In some initial experiments, chloroform was used instead of diethyl ether to remove MeOH. For data from the TU8988T cells, the aqueous-methanol extracts after addition of chloroform were directly evaporated to dryness in miVac centrifugal evaporator (Genevac) without applied heat. The final extracts were stored at −80°C typically for 1–3 days before the UDP-GlcNAc measurements.
10
Identifying Novel ARX Interacting Proteins
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The whole embryonic forebrains (E14.5) were triturated in buffer B (25 mM Tris-Cl, pH 7.4, 5% glycerol, 3 mM KCl, 140 mM NaCl, 1% Triton X-100, 0.2 mM EDTA, 1.5 mM MgCl2, protease and phosphatase inhibitors; Roche Biochem), and sonicated on ice (Branson digital sonifier 250, 5 cycles of sonication at 2 sec “On” and 2 sec “Off’ at 10% output). The crude lysates were centrifuged at 13,000 x g for 15 min at 4°C, and the supernatants were pre-cleared with protein A/G agarose bead for 1 hr at 4°C with rotation. In order to isolate novel ARX-binding proteins, ARX antibody against amino acids 158–181 was generated in rabbit and immobilized on Dynabeads M-280 Tosylactivaed (Invitrogen) according to the manufacturer’s instructions, and the beads were incubated with the pre-cleared whole embryonic brain lysate for 2 hr at 4°C with rotation. The beads were washed in buffer B three times (each time for 3 min at 4°C with rotation), and bound protein were acid-eluted and immediately neutralized. The eluted proteins were analyzed at the Keck Mass Spec & Proteomics Resource (Yale School of Medicine) and Proteomics & Systems Biology core (Perelman School of Medicine at the University of Pennsylvania).
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