All steps were performed on ice. Cells were scraped in cold PBS completed with 100 mM N-ethylmaleimide (NEM, Sigma Aldrich, Saint Louis, MO, USA), transferred into pre-chilled 1.5 mL tubes and pelleted by centrifugation at 16,200× g for 20 s at 4 °C. Pellets were immediately resuspended in 70 μL cold PBS supplemented with 100 mM NEM, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 5 mM NaF, 1 mM activated Na3VO4, 2 mM p-nitrophenyl phosphate, and 10 mM β-glycerophosphate (Buffer I). Samples were sonicated using 3 × 15 pulses of 0.8 s at 40% (UP50H sonicator, Hielscher) with rounds on ice in between. After centrifugation at 800× g for 10 min at 4 °C, the supernatants were transferred into a clean pre-chilled 1.5 mL tube before adding an equal volume of cold Buffer I supplemented with 0.1% SDS. Samples were incubated on ice for 15 min and quantified using the BCA method (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA). Freezing of samples was found to be detrimental to the detection of aggregates; therefore, WCEs were analyzed immediately by SDD-AGE or non-reducing SDS-PAGE as described below.
Up50h sonicator
Manufactured by Hielscher
Sourced in Germany
The UP50H sonicator is a high-performance ultrasonic homogenizer designed for laboratory use. Its core function is to disrupt and disperse samples through the application of high-frequency sound waves, enabling efficient sample preparation and extraction procedures.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Anti erk1 2, by Santa Cruz Biotechnology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti rat hrp, by Jackson ImmunoResearch (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Simplechip enzymatic chromatin ip kit with agarose beads, by Cell Signaling Technology (1 mentions)
Acetone, by Thermo Fisher Scientific (1 mentions)
8 protocols using up50h sonicator
1
Extraction and Analysis of Aggregated Proteins
2
Purification of GST-fusion Proteins
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The vectors pGEX-4T3, pGEX-4T3-ASPG and pGEX-4T3-ASPG (T19A) were used to transform E.coli BL21 cells. Bacteria were exponentially grown at 37°C in LB medium supplemented with 100 μg/mL ampicillin and subsequently induced to express fusion proteins by 0.5 mM isopropyl-thiogalactopiranoside (IPTG) for 3h at 37°C. Cells were then centrifuged at 4.000 x g for 15 min, washed with 1X cold phosphate-buffered saline (PBS), resuspended with lysis buffer containing 1% triton X-100, 1% glycerol, 10% sarkosyl, 4 mg/mL lysozyme, 20 mM CHAPS, 10 mM NaF, 2 mM orthovanadate in cold PBS 1X supplemented with protease inhibitors (Roche Molecular Biochemicals) and finally sonicated three times for 30 seconds using a Hielscher UP50H sonicator (0.5 cycles, amplitude 100). Fusion proteins were purified from crude E.coli extract by single-step affinity chromatography using Glutathione Sepharose® 4B-beads (GE Healthcare) according to the manufacturer's instructions and finally dialyzed at 4°C with 1 mM EDTA, 50 mM NaCl, 50 mM Tris/HCl pH 8 buffer. The concentration of each purified GST-fusion protein was estimated by Coomassie Brilliant Blue R-250 staining using a standard curve of BSA.
3
Immunoblotting of Phosphorylated ERK1/2
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Frozen cell pellets were dissolved in Laemmlie-buffer (60 mM Tris-HCl, 10% glycerol, 2% SDS, 10% β-mercaptoethanol, 0.01% bromophenol blue; pH 6.8) (10 µl buffer per 100,000 cells) and subjected to sonication (3–5 s on ice with a UP50H sonicator equipped with an MS1 sonotrode) (Hielscher, Teltow, Germany). Samples were then heated to 89°C for 3 min, spun for 5 min at room temperature and the supernatants used for standard SDS-PAGE with 12% gels. Wet blotting was carried out in Mini Trans-Blot modules (Bio-Rad Laboratories) using nitrocellulose membranes and blotting buffer (20% v/v methanol, 25 mM Tris-HCl, 192 mM glycine, pH 8.6). The following antibodies were used for target detection: anti-ERK1/2 (Cell Signaling Technology, Frankfurt am Main, Germany; no. 9102), anti-ERK1/2 (Santa Cruz Biotechnology, Heidelberg, Germany; sc-94), anti-phospho-ERK1/2 (Cell Signaling Technology; no. 9101), anti-tubulin (Biozol, Eching, Germany; BZL03568). The secondary antibodies were from Jackson ImmunoResearch Laboratories (Newmarket, UK): anti-rat-HRP (112-036-062), anti-rabbit HRP (111-036-045). A freshly made solution of luminol (2.5 mM), p-coumaric acid (0.2 mM) and H2O2 (0.01%) in 100 mM Tris-HCl (pH 8.8) was used for chemiluminescent detection [29] (link).
4
Catabolic Myotube Culture Protocol
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C2C12 and fa‐C2C12 myoblasts were grown under 5% CO2 in Dulbecco's modified Eagle medium (DMEM, Sigma‐Aldrich, Saint‐Quentin Fallavier, France) containing 10% foetal bovine serum (FBS, Gibco, Invitrogen, Paislay, UK) and supplemented with l ‐glutamine, non essential amino acids, and gentamycin (Gibco, growth medium, GM). For myoblast differentiation, cells were grown to near confluence and then shifted to DMEM containing 2% horse serum (differentiation medium) for 6 days.
A catabolic state was induced in myotubes by adding Dex up to 48 h before harvesting the cells. Briefly, myotubes were washed and scraped off the plate into 1X phosphate‐buffered saline and then sonicated for 30 s at maximum power using a UP50H sonicator (Hielscher, Teltow, Germany) in lysis buffer (5 mM Tris, pH 7.5, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM NEM, 1% Triton X‐100/anti‐proteases (Protease Inhibitor Mixture/Sigma)) as previously described.28 Cell lysate was then centrifuged at 10 000 g for 10 min at 4 °C, and the supernatant (soluble fraction) was kept at −80 °C until use. The pellets enriched in myofibrillar proteins were resuspended in homogenization buffer and sonicated to re‐suspend the proteins. Protein content was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA).
A catabolic state was induced in myotubes by adding Dex up to 48 h before harvesting the cells. Briefly, myotubes were washed and scraped off the plate into 1X phosphate‐buffered saline and then sonicated for 30 s at maximum power using a UP50H sonicator (Hielscher, Teltow, Germany) in lysis buffer (5 mM Tris, pH 7.5, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM NEM, 1% Triton X‐100/anti‐proteases (Protease Inhibitor Mixture/Sigma)) as previously described.
5
ChIP Assay for Histone Acetylation
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ChIP assays were performed using the SimpleChiP® Enzymatic Chromatin IP Kit with Agarose beads (Cell Signaling). In brief, proteins were cross-linked to DNA with 1.5% formaldehyde for 10 minutes. Nuclear membranes were broken up using the UP50H Sonicator (Hielscher, Teltow, Germany) set to 100%, 0.9 output for 20 seconds, 6 times in a row with incubation on ice for 30 seconds between sonication pulses. Afterwards, antibodies against histone H3 (cat. no. 6420), acetyl-histone H3 (Lys9) (AcH3K9) (cat. no. 9671), or normal rabbit IgG (Cell Signaling) were used for immune precipitation. The immune precipitated DNA was subsequently analyzed by qRT-PCR, using SYBR green primers specific to the MICA or MICB promoter region: MICA promoter sense CGG ATC CTG GAA TAC GTG GG, antisense ACT CAC ACC TGC CCG TTA TG; MICB promoter sense GCG ACA GGG TCC AGG TCG TGC TC, antisense CCC TAC GTC GCC ACC TTC TCA GCT. The percentage of acetylated histones (AcH3K9) was normalized to total Histones H3 and calculated using following equation:
6
Protein Extraction from Lens Capsules
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Each lens capsule sample was sonicated for 30 sec (using the UP50H sonicator by Hielscher Ultrasonics GmbH, Teltow, Germany, connected with a 2-mm diameter MS2 microtip) in the presence of 92 μl double-distilled water (ddΗ2Ο) and 8 μl 1% DOC stock solution (made in ddΗ2Ο). Then, the sonicated homogenate was mixed with 300 μl ddΗ2Ο, and the mixture was centrifuged at 16,000 ×g for 5 min at 4 °C to remove any insoluble material. The resulting supernatant was incubated for 10 min at 25 °C, followed by the addition of 46 μl ice-cold 100% TCA (final 10% TCA, 0.019% DOC), incubation for 15 min in an ice-water bath, and centrifugation at 16,000 ×g for 5 min at 4 °C. This DOC-TCA protein precipitation procedure is a modification of a procedure previously reported [87 (link)-89 (link)], which is able to precipitate minute quantities of proteins (as low as 3 µg) with ≥90% recovery. The resulting protein pellet was dissolved in 50 µl 50 mM NaOH containing 4 M urea and stored at −80 °C for measuring protein carbonyls and protein concentration.
7
Intestinal Tissue Homogenization and Protein Quantification
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Fragments of 0.1 g from intestine tissue were mixed with 1 mL of ice-cold TRIS-EDTA buffer (0.1 M TRIS-HCl buffer containing 5 mM EDTA, pH 7.4) and homogenized (seven times, 30 s each) on ice using a UP 50H sonicator (Hielscher). After 30 min centrifugation of tissue homogenates at 10,000 rpm, 4 °C, the resulting supernatants were collected and preserved at −80 °C for further analyses. The total protein concentration was measured according to the method described by Lowry [26 (link)] using 0.01 g mL−1 bovine serum albumin (BSA) as standard.
8
Thiol Redox Proteome Profiling
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Unless otherwise stated, all steps were performed on ice and in the dark. At time of harvesting, cells were immediately transferred to pre-chilled tubes and washed in cold ddPBS before trichloroacetic acid (TCA, Sigma Aldrich)/acetone (Fisher chemical) precipitation (11) . After TCA/acetone addition, samples were sonicated (3x15 pulses, 70%, UP50H sonicator, Hielscher) with rounds on ice. Samples were stored at -80°C for at least 2h before further processing. The bioswitch labeling procedure was performed according to the protocols described in (11, 45) with modifications using N-ethylmaleimide (NEM, Sigma Aldrich) to alkylate the reduced thiols, tris(2-carboxyethyl)phosphine (TCEP, EMD Millipore Sigma) to reduce the oxidized thiols and EZ-Link Maleimide-PEG 2 -Biotin (Mal-PEG 2 -Bio, Thermofisher) to label the newly reduced thiols (Fig. 1A). Briefly, 12mg of TCA/acetone precipitated proteins were used for alkylation with 3mM NEM in 0.5M Tris , 1% SDS (BioShop) pH 7.3 for 30min at room temperature (RT). Next, reduction was performed using 4mM TCEP in 0.5M Tris , 1% SDS pH 7.0 for 1h at RT followed by labeling with 0.25mM Mal-PEG 2 -Bio for 2h at RT.
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