For immunoblotting analysis, BMSCs incubated with 50 ng ml–1 IL-17 for 15, 30, and 60 min or 10 ng ml–1 TNFα for 7.5, 15, 30 min were pre-challenged by 100 nM RA for 6 h. Cells were washed with ice-cold PBS, harvested and lysed for 15 min by lysis buffer containing 0.5% TritonX-100 (T9284, Sigma), 20 mM Hepes pH7.4 (H-4034, Sigma), 150 mM NaCl (A100241, Sangon Biotech), 12.5 mM β-glycerophosphate (A500486, Sangon Biotech), 1.5 mM MgCl2 (M4880, Sigma), 2 mM EGTA (A600077, Sangon Biotech), and a cocktail of protease inhibitors, Na3VO4 (A600869, Sangon Biotech), NaF (A500850, Sangon Biotech), and PMSF (A610425, Sangon Biotech). Equal amounts of protein extracts were resolved in 10% SDS-PAGE and transferred to PVDF membranes (IPVH00010, Merck Millipore). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST, pH 7.6) for 1 h at room temperature before incubated overnight with the primary antibodies (p65 1:1,000, pp65 (Ser536) 1:1,000, IκBα 1:1,000, pIκBα (Ser32) 1:1,000, β-actin 1:1,000) at 4°C and then incubated with the secondary antibodies (rabbit, 1:10,000, W401B, Promega) for 1 h at room temperature. Finally, the blots were detected by enhanced chemiluminescent reagents (Millipore).
H4034
Manufactured by Merck Group
Sourced in United States
H4034 is a laboratory equipment product manufactured by Merck Group. It is designed for various scientific applications within research and development settings. The core function of H4034 is to provide a controlled environment for conducting experiments and analyses. Further details on the intended use or specific applications of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
No69989, by Qiagen (2 mentions)
Thermomixer, by Eppendorf (2 mentions)
Tissuelyzer, by Qiagen (2 mentions)
U6253, by Merck Group (2 mentions)
Ed 100, by Merck Group (2 mentions)
S0389, by Merck Group (2 mentions)
Enhanced chemiluminescent reagent, by Merck Group (1 mentions)
T9284, by Merck Group (1 mentions)
M4880, by Merck Group (1 mentions)
A610425, by Sangon (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Millicell cm, by Merck Group (1 mentions)
Np 40, by Merck Group (1 mentions)
Odyssey, by Bio-Rad (1 mentions)
Clarity western ecl substrate solution, by Bio-Rad (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Odyssey infrared scanner, by LI COR (1 mentions)
Irdye 680, by LI COR (1 mentions)
Irdye 800, by LI COR (1 mentions)
14 protocols using h4034
1
Immunoblotting Analysis of BMSCs under IL-17 and TNFα
2
Metabolite Profiling of PDX and Clinical Samples
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LC‐MS analysis was run on snap‐frozen tissue of six PDX samples and 13 clinical samples. Each sample was analyzed in three replicates. For metabolite extraction, a 5‐mm metal bead (Qiagen, No69989) and extraction buffer (methanol/acetonitrile/water‐50/30/20) containing 100 ng/ml HEPES for internal standard purpose (Sigma, H4034) were added to the tissue at a volume/weight ratio of 25 μl/mg. The tissue was disrupted in a bead mill (Qiagen, Tissuelyzer, Hombrechtikan, Switzerland) with 2 cycles (2 × 20 s/20 MHz) before vortexing on an Eppendorf Thermomixer at 2°C/20 min/1,400 rpm. The extract was clarified by centrifugation (15 min/13,362 g/4°C in an Eppendorf centrifuge) and transferred in fresh tubes and stored at −80°C. LC‐MS analysis was performed as previously described (Tardito et al, 2015 ). Tumor extracts were compared to extracts from contralateral mouse brain for reliable comparison between the two models.
3
Hippocampal Slice Culture Protocol
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Seven-day-old Sprague-Dawley rats were sacrificed and their brains were quickly extracted and immersed in ice-cold HBSS medium (LB 003-01, Sigma, St. Louis, Mo, USA) with 20 mM HEPES (H-4034, Sigma, St. Louis, Mo, USA) immediately. The cerebellum and frontal cortex were removed delicately and the hippocampus was extracted and amputated transversely at 350 µm by a tissue chopper (Mickle Laboratory Engineering Co., Surrey, UK). In each six-well plate, four or five slices were placed onto a 0.4 µm culture insert (Millicell-CM; Millipore, Bedford, MA, USA). The six-well plate was filled with 1mL of culture medium (MEM; 25%(v/v) Hank's balanced salt solution, supplemented with 20 mM HEPES, 6 g/L D-glucose, 1 mM L-glutamine, 25%(v/v) heat-inactivated horse serum and 1% penicillin-streptomycin; pH=7.1). The culture was incubated at 36℃ with 5% CO2 and 95% O2. The culture medium was replaced three times a week and the sections were cultured for two weeks [4 (link)].
4
In vitro ubiquitination assay
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A method for in vitro ubiquitination assays is described elsewhere (Asaoka et al., 2016 (link); Fennell et al., 2020 (link)). Ubiquitin (10 μg) (Sigma-Aldrich, U6253), mouse E1 (Ube1) (150 ng), human Ubch7 (300 ng), human HOIP (5 μg), human SHARPIN (1μg), human HOIL-1L (1 μg), human NEMO (5 μg) and ATP (2 mM) (Roche, 10519979000) were incubated at 37°C for the indicated time in a buffer containing 150 mM NaCl, 20 mM MgCl2 and 50 mM HEPES (Sigma Aldrich, H4034) (pH7.5). Reactions were terminated by adding SDS sample buffer and incubation at 96°C for 1 minute. Samples were subjected to SDS-PAGE followed by immunoblotting.
5
Western Blot Protein Analysis
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Cells were washed with ice-cold PBS and lysed with 25 mM Hepes, pH 7.2 (H4034; Sigma-Aldrich), 125 mM potassium acetate (104820; Merck Millipore), 2.5 mM magnesium acetate (105819; Merck Millipore), 5 mM EGTA (E3889; Sigma-Aldrich), 0.5% NP-40 (I18896; Sigma-Aldrich), and 1 mM DTT (D0632; Sigma-Aldrich) supplemented with protease inhibitor cocktail (P9340; Sigma-Aldrich). Cell lysates were then subjected to SDS-PAGE on 10% (567-1034; Bio-Rad) or 4%–20% (567-1094; Bio-Rad) gradient gels and blotted onto Immobilon-P membranes (IPVH00010; Merck Millipore). Membranes incubated with fluorescently labeled secondary antibodies (IRDye680 and IRDye800; LI-COR) were developed by Odyssey infrared scanner (LI-COR). Membranes detected with HRP-labeled secondary antibodies were developed using Clarity Western ECL substrate solutions (Bio-Rad) with a ChemiDoc XRS+ imaging system (Bio-Rad). Quantification of immunoblots was performed using the Odyssey, Volume tools of Image Lab (Bio-Rad), or ImageJ software (National Institutes of Health).
6
Subcellular Fractionation by Differential Centrifugation
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Cells, rinsed in ice-cold PBS and centrifuged at 800 × g for 10 min, were resuspended in homogenization buffer (HB: 0.25 M sucrose (Sigma-Aldrich, S0389), 10 mM HEPES pH 7 (Sigma-Aldrich, H4034), EDTA (Sigma-Aldrich, ED-100)) with protease and phosphatase inhibitors cocktail. Nuclei, mitochondria and cytosol purifications proceed by standard differential centrifugations. After homogenization of the solution with 30–70 pulses (one pulse corresponds to approximately 1 s), cells were collected at 600 × g at 4 °C for 10 min. The pellet containing the nuclear fraction was suspended in RIPA buffer, instead the supernatant was subjected to a further centrifugation at 11,000 × g for 15 min at 4 °C. The mitochondrial pellet fraction was suspended in RIPA buffer. The supernatant fraction corresponded to soluble cytosolic proteins.
7
Mitochondria and Cytosol Purification
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Mitochondria and cytosol purification occurs by standard differential centrifugations. HEK293 cells were suspended in homogenization buffer (0.25 M sucrose, 10 mM HEPES, pH 7, 1 mM EDTA [Sigma-Aldrich, S0389, H4034, and ED-100, respectively]) with protease and phosphatase inhibitors. Mitochondria suspensions were obtained homogenizing the solution with 40°–60 pulses (one pulse corresponds to approximately one sec), in vertical movements. After homogenization, the suspension was centrifuged at 11 000 g for 15 min at 4°C. The mitochondrial pellet fraction was suspended in isolation buffer (0.2 M sucrose, 10 mM Tris-MOPS [Sigma, M1254], pH 7.4, 0.1 mM EGTA-Tris [Sigma Aldrich, E4378]). The supernatant fraction corresponded to soluble cytosolic proteins.
8
Tissue Metabolite Extraction Protocol
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The collected tissue fragments were subdivided on ice in smaller fragments (10–20 mg) and transferred in 2 ml Eppendorf tubes for metabolite or nucleic acid extractions. For metabolite extraction, a 5 mm metal bead (Qiagen, No69989) and extraction buffer (methanol/acetonitrile/water-50/30/20) containing 100 ng/ml HEPES for internal standard purpose (Sigma, H4034) was added to the tissue at a volume/weight ratio of 25 μl/mg. The tissue was disrupted in a bead mill (Qiagen, Tissuelyzer, Hombrechtikan, Switzerland) with two cycles (2 min/20 MHz) before vortexing on an Eppendorf Thermomixer at 2 °C/20 min/1,400 rpm. The extract was clarified by centrifugation (15 min/12k rpm/4 °C in an Eppendorf centrifuge) and transferred into fresh tubes for storage at −80 °C for LC–MS.
9
Collagen-Based Contractility Assay for ASCs
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Type I rat tail collagen (Corning, 354236) was diluted and neutralized in an equal volume of filter sterilized HEPES neutralization solution (2× PBS pH = 7.4, 0.1 M HEPES pH = 7.4, Sigma H4034). A total of 5×104 CD and HFD ASCs were trypsinized, counted, and added to the collagen to bring the final concentration to 1 mg/ml. The neutralized collagen and cell mixture was plated on six-well plates and incubated at 37°C. After 4 hours, the gels were released and floated in 2 ml of DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic solution. The gel diameter was measured with a ruler at the time of gel release at day 0 and on days 1, 3, 5, and 6. Gels were fed after measurement on day 3. Gels were digested with collagenase (Roche, 11088793001) for 10 minutes at 37°C, and ASCs were counted to normalize the contracted area of the gel to the number of total cells on day 6. The assay was run in triplicate with three biological replicates for both CD and HFD ASCs. Contracted area was calculated using A = πr2 and subtracting the area from the beginning area at day 0. The difference in area of contraction was divided by the number of cells in the gel at day 6.
10
Western Blot Analysis of Ube3a Proteins
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Western blot analysis was carried out for AS (Ube3ap+/m−) and WT (Ube3ap+/m+). The cells were lysed with an ice-cold lysis buffer containing: 10 mM HEPES pH 7.5 (Sigma-Aldrich #H4034), 150 mM NaCl (Sigma-Aldrich #71376), 50 mM NaF (Sigma-Aldrich #201154), 1 mM EDTA (Sigma-Aldrich #ED), 1 mM EGTA, 10 mM Na4P2O7 (Sigma-Aldrich #P8010) PMSF, and protease inhibitor cocktail (Roche #11873580001). Cell lysates were loaded (15–20 µg per lane) to 4–20% TEO TRICINE gradient gels (abcam ab119209) and transferred to PVDF membranes (abcam ab133411). The membrane was blocked in 5% bovine serum albumin (BSA; Roche #10735086001) in tris-buffered saline with tween20 (TBST) before incubation with primary antibodies for UBE3A (anti-mouse; 1:1000; Sigma-Aldrich #E8655), BCL-2 (anti-rabbit, 1:2000, abcam ab182858), BAX (anti-rabbit, 1:2000, abcam ab182733) overnight. β-ACTIN (anti-mouse; 1:40,000; MP Biomedicals #69100) was used as a loading control. Next, the blots were incubated with the secondary antibodies goat anti-mouse or anti-rabbit IgG (H + L) (1:10000; Jackson #115–035–062 and #115-035-068, respectively). The bands were detected by chemiluminescence (abcam ab133406) and imaged using the Image Quant LAS 4000 system. All signals were normalized by the total protein and quantified using Image Studio Lite Ver 5.2 software.
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