Cells were collected in lysis buffer (50 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.5% Triton X-100) supplemented with the Pierce Protease and Phosphatase Inhibitor tablets (Thermo Scientific) and sonicated. DynabeadsTM M-280 Sheep Anti-Mouse IgG (Life Technologies) were washed in 3% BSA/PBS. GFP or p62 antibodies were coupled to Dynabeads (1 µg antibody/50 µl beads), by incubating with rotation overnight at 4 °C. Cell lysates were precleared with 5 μL Dynabeads for 1 hour at 4 °C and then incubated with antibody-Dynabeads with rotation overnight at 4 °C. The immunoprecipitates were washed in 3% BSA/PBS and then in PBS, and further transferred to a clean tube. The proteins were eluted by boiling in 1x laemmli buffer at 90 °C for 10 min.
Pierce protease and phosphatase inhibitor tablet
Manufactured by Thermo Fisher Scientific
Sourced in United States
Pierce protease and phosphatase inhibitor tablets are used to inhibit the activity of proteases and phosphatases in biological samples. They are designed to help preserve the integrity of proteins during sample preparation and analysis.
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Lab products found in correlation
Nupage 4 12 precast gels, by Thermo Fisher Scientific (2 mentions)
Odyssey infrared scanning system, by LI COR (2 mentions)
Immobilon fl, by Merck Group (2 mentions)
Immobilon p transfer membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Lonza (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Cytiva (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Medical x ray processor 102, by Kodak (1 mentions)
β actin, by Fortis Life Sciences (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Apoptosis western blot cocktail, by Abcam (1 mentions)
Anti β actin antibody, by Fortis Life Sciences (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent, by Thermo Fisher Scientific (1 mentions)
10 protocols using pierce protease and phosphatase inhibitor tablet
1
Immunoprecipitation of GFP and p62 Proteins
2
Western Blot Protein Analysis
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For Western blotting, cells were lysed in PBS with 2% Triton X-100 containing Pierce protease and phosphatase inhibitor tablets (Thermo Fisher Scientific). The protein concentration was determined with a bicinchoninic acid assay kit (Thermo Fisher Scientific), and equal amounts were resolved on NuPAGE 4–12% precast gels (Invitrogen). Blotting was performed on polyvinylidene fluoride membranes (Immobilon-FL, EMD Millipore) followed by detection using the Odyssey infrared scanning system (LI-COR Biosciences).
3
Anti-inflammatory effects of AJHLE
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This was performed with available commercial kits using the enzyme-linked immune sorbent assay (ELISA) technique. In fact, murine RAW 264.7 cells were seeded at a density of 2 × 105 cells per well into 12-well plates, and they were allowed to attach overnight. These cells were pre-treated with DMSO 0.5% (negative control), quercetin at 25 µM (positive control), and AJHLE at different concentrations (12.5, 25, and 50 µg/mL) for 2 h, followed by the addition of LPS (200 ng/mL) for 24 h under standard cell-culture conditions. Thereafter, treated cells were washed twice with pre-cooled phosphate-buffered saline (PBS) (Cytiva Hyclone, Logan, UT, USA) and dissociated using trypsin-EDTA (Cytiva Hyclone, Logan, UT, USA). The cell lysates were obtained in PBS containing EDTA-free Pierce Protease and Phosphatase-inhibitor Tablets (ThermoFisher Scientific, Lenexa, KS, USA) by repeating freeze–thaw cycles in liquid nitrogen and a water bath (37 °C). After centrifugation at 10,000 rpm at 4 °C, the cell supernatants were collected and used for the quantification of cyclooxygenase-2 (COX-2), interleukins (IL-1β and IL-10), and tumor necrosis factor (TNF-α) concentrations using Elabscience® Mouse COX-2, IL-1β, IL-10, and TNF-α ELISA kits (Elabscience Biotechnology Inc., Houston, TX, USA) according to the manufacturer’s instructions.
4
Western Blotting Protein Detection
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For western blotting, cells were lysed in PBS containing 2% Triton X-100 and Pierce protease and phosphatase inhibitor tablets (Thermo Fisher Scientific). The protein concentration was determined with a BCA assay kit (Thermo Fisher Scientific), and equal amounts were resolved on NuPAGE 4–12% precast gels (Invitrogen). Proteins were transferred from gels onto PVDF membrane (Immobilon-FL, pore size 0.45 μm, Millipore, catalogue number IPFL00010) for immunoblotting. Transfer was performed in transfer buffer (25 mM Tris, 192 mM glycine and 10% methanol) at 100 V for 70 min. Membranes were blocked in 5% milk in PBS containing 0.1% Tween 20 (PBS-T) prior to incubation with the appropriate primary antibodies and fluorescently labelled secondary antibodies. Detection and quantification were carried out using an Odyssey infrared scanning system (LI-COR Biosciences).
5
Molecular Profiling of Ndufs4 Mouse Brain
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Vehicle-treated Ndufs4+/+ and Ndufs4−/− mice, or ruboxistaurin- or rapamycin-treated Ndufs4+/+ and Ndufs4−/− mice were treated daily from P10 until P27–31 or P47–51. Mice were then fasted overnight (for 12 h), treated with vehicle or small molecule, and re-fed. Unanesthetized mice were then euthanized by cervical dislocation 3–4.5 hours later. Brains were immediately isolated and flash frozen in liquid N2 for Western blot. Brains were ground using a cryogenic homogenizer. Approximately 50 mg of homogenized brain tissue was lysed with 1 mL RIPA buffer containing Pierce™ protease and phosphatase inhibitor tablets (Thermo Fisher) for 30 minutes. Samples were then centrifuged to remove insoluble cell debris, quantified by BCA, diluted to 2 mg/mL, and used for Western blot.
6
Molecular Profiling of Ndufs4 Mouse Brain
7
Western Blot Analysis of GIST Cells
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Cultured GIST cell lines were rinsed in PBS (Lonza) and scraped on ice into a RIPA lysis buffer (Pierce Biotechnology, Rockford, IL, USA) containing a Pierce Protease and Phosphatase Inhibitor Tablet (Thermo Fisher Scientific Inc.), followed by sonication. The protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Ten microgram of protein was separated using gel electrophoresis and blotted onto an Immobilon‐P Transfer Membrane (Millipore, Billerica, MA, USA). The primary antibodies were a monoclonal rabbit anti‐ITGA4 antibody (dilution 1:1000; clone D2E1; Cell Signaling Technology, Danvers, MA, USA), a polyclonal rabbit anti‐c‐KIT antibody (dilution 1:10,000; A4502; Agilent Technologies Dako, Glostrup, Denmark), a polyclonal rabbit anti‐Phospho‐c‐KIT (Tyr703) antibody (dilution 1:10,000; 3391; Cell Signaling Technology), a polyclonal rabbit β‐Actin (dilution 1:10,000; Bethyl Laboratories, Montgomery, TX, USA) and a peroxidase‐conjugated AffiniPure Goat Anti‐Rabbit antibody was used as the secondary antibody (dilution 1:10,000; Jackson Immuno Research, West Grove, PA, USA). Blot immunostains were treated with a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.), exposed to an X‐ray film, and the films were developed with a Kodak Medical X‐ray Processor 102 (Eastman Kodak; Rochester, NY, USA).
8
Comparative Analysis of c-KIT and SLUG Expression in GIST Cell Lines
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Cultured GIST882 and GIST48 cells were rinsed in PBS (Lonza, Walkersville, MD, USA) and scraped into a RIPA lysis buffer (Pierce Biotechnology, Rockford, IL, USA) containing a Pierce Protease and Phosphatase Inhibitor Tablet (Thermo Fisher Scientific Inc., Rockford, IL, USA), followed by sonication. Protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Protein 10 μg of protein was separated using a gel electrophoresis and blotted onto an Immobilon-P Transfer Membrane (Millipore, Billerica, MA, USA), and stained with a polyclonal rabbit anti-c-KIT antibody (dilution 1 : 10000; clone A4502, Agilent Technologies Dako, Glostrup, Denmark), a polyclonal rabbit anti-SLUG antibody (dilution 1 : 1000; clone C19G7, Cell Signaling Technology), a polyclonal rabbit anti-β-actin antibody (dilution 1 : 10 000; Bethyl Laboratories, Montgomery, TX, USA) or a Apoptosis Western Blot Cocktail (Abcam, Cambridge, UK). Primary antibodies were detected with specific horseradish-labelled secondary antibodies and using the SuperSignal West Pico and Femto Chemiluminescent Substrate (Thermo Fisher Scientific Inc.), following manufacturer's instructions. The basal level of SLUG expression was significantly lower in GIST882 cells than in GIST48 cells. Protein expression was detected after diluting Femto Substrate 1 : 5 with Pico Substrate.
9
Immunoprecipitation and Western Blot
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Confluent cells were washed with PBS and lysed with RIPA buffer (10 mM Na2HPO4, 150 mM NaCl, 5 mM EDTA, 2 mM EGTA, 1% Triton X‐100, 0.25% SDS, 1% sodium deoxycholate, 0.1 mM DTT, pH 7.5) supplemented with Pierce protease and phosphatase inhibitor tablet (Thermo Fisher Scientific). Lysates were incubated for 30 min at 4°C and centrifuged at 16,000×g for 10 min at 4°C. Supernatants were pre‐cleared with 30 µL Dynabeads Protein G (Thermo Fisher Scientific) and incubated for 1 h at 4°C. The specific antibody or IgG control was added to cell lysates at a concentration of 1 µg antibody per 0.5 mg protein lysate and incubated overnight at 4°C. Dynabeads Protein G were added, and the samples were incubated under rotation at 4°C for 3 h. After several washing steps, bead‐bound proteins were eluted by addition of sample buffer, heated for 15 min at 95°C and analysed by western blotting.
10
Protein Extraction and Western Blot Analysis
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The harvested cells were lysed with M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) that containing Pierce Protease and Phosphatase Inhibitor Tablet (Thermo Fisher Scientific, USA). Then, samples (20 μg protein/lane) were electrophoresed by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto PVDF membrane. The membrane was blocked with Western Blocking Buffer (CWBIO, China) for 1 h at room temperature and subsequently incubated overnight at 4°C with anti-p21/ CDKN1A (1:1000; CST, #2947), anti-p27/CDKN1B (1:1000; CST, #3686), anti-Akt (1:1000; CST, #4691), anti-p-Akt (Thr308) (1:1000; CST, #13038), anti-p-Akt (Ser473) (1:1000; CST, #4060) and anti-β-actin (1:1000; CST, #4970). Then, the membrane was incubated with a horseradish peroxidase-conjugated goatanti-rabbit IgG (1:10000; Millipore，#AP307P, USA) for 1 h. Chemiluminescence was detected using the SuperSignal West pico chemiluminescent (Thermo Fisher Scientific, USA).
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