After HUVECs or mouse lung tissue were lysed in lysis buffer (R&D Systems, Minneapolis, MN, USA), each solubilized protein sample was adjusted to an equal concentration. The levels of inflammatory cytokines, namely interleukin 1α (IL)-1α and IL-6, and growth factors, such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), were measured using commercial ELISA kits (R&D System), according to the manufacturer’s protocol.
Lysis buffer
Manufactured by R&D Systems
Sourced in United States
Lysis buffer is a solution used to break down and disrupt the cell membrane and release the cellular contents, including proteins, nucleic acids, and other cellular components. It is a core component in various cell biology and molecular biology protocols, enabling the extraction and isolation of these biomolecules for further analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Multi well reader, by Thermo Fisher Scientific (3 mentions)
Hdac activity assay kit, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
60 mm culture dish, by BD (2 mentions)
96 well microtiter plate, by SPL Life Sciences (2 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Hdac assay kit, by Merck Group (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Human protease array kit, by R&D Systems (1 mentions)
Odyssey fc dual mode imaging system, by LI COR (1 mentions)
Amersham protran, by GE Healthcare (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Multi wall reader, by Thermo Fisher Scientific (1 mentions)
Human survivin quantikine elisa kit, by R&D Systems (1 mentions)
Chemi reagent mix, by R&D Systems (1 mentions)
Human apoptosis proteome profiler array, by R&D Systems (1 mentions)
Staurosporine, by Merck Group (1 mentions)
Biotek cytation 5 system, by Agilent Technologies (1 mentions)
β actin, by Abcam (1 mentions)
Protease inhibitor, by Roche (1 mentions)
25 protocols using lysis buffer
1
Cytokine and Growth Factor Quantification
2
Assaying Histone Deacetylase Activity in HeLa Cells
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Histone deacetylase activity was assayed, as described [93 (link)]. HeLa cells were treated with TSA, PdNPs or in combination for 24 h. The cells were washed in PBS and suspended in 5 volumes of lysis buffer (R&D Systems, Inc., Minneapolis, MN, USA). Next, cells were harvested, and whole cell protein was extracted, using RIPA lysis buffer. Protein concentrations were measured, using BCA kit reagents. Supernatant samples, containing 20 µg of total protein, were used to assay HDAC activity. The samples and HDAC substrate, provided by the assay kit, were added to each well of a 96-well microtiter plate and incubated at 37 °C for 1 h. HDAC activity was measured using the HDAC Activity Assay kit (Sigma-Alrich, St. Louis, MO, USA). Experimental procedures were performed, according to the manufacturer’s instructions.
3
Kinase Pathway Profiling in CRC Cells
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CRC cell lines were treated as described and washed in 1X PBS. Cells were solubilized using lysis buffer provided by the vendor (R&D Systems) and rocked for 30 min at 4 °C. Suspension was spun for 5 min at 14,000 × g and supernatant was collected. Concentration of protein in the collected supernatant fluid determined using the BCA assay (ThermoFisher Scientific, no. 23227). 200 μg of sample lysate was applied to nitrocellulose membranes kinase arrays and incubated overnight at 4 °C. Provided detection antibodies were incubated with specified concentrations as suggested by the supplier. Membrane arrays were acquired using Odyssey Infrared Imaging System (LI-COR Biosciences) and quantified by densitometry using NIH ImageJ software. Values from densitometry analysis were normalized to HSP60 control. Normalized value was then converted to Log2 fold change and plotted on heatmap using Graphpad Prism 8.
4
Quantification of HDAC Activity
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The HDAC activity was measured by using the HDAC assay kit, according to manufacturer's instructions (Millipore, Billerica, MA). Briefly, 1 × 106 cells in 60 mm culture dish (BD Falcon) were incubated with or without 5 μM SAHA for 24 hours. Then cells were washed with PBS and added in 4 volumes of lysis buffer (R&D systems, Inc.). Thirty μg of total protein were used to measure the HDAC activity. These were added to each well in 96-well microtiter plates (SPL Life Sciences, Pocheon, Gyeonggi-do, Korea) with HDAC substrate provided from the kit at 37°C for 1 hour. The optical density of each well was measured at 405 nm by using a microplate reader (Synergy™ 2, BioTekR Instruments Inc., Winooski, VT).
5
Immunoprecipitation of OLA1 Protein
6
Measuring Phospho-ERK1/2 in Jurkat T Cells
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Jurkat T cells were incubated for 20 min with the selected compounds or negative control (1% DMSO) at 37 °C, followed by addition of anti-CD3/C28 monoclonal antibodies (10 µg/mL of each antibody) and a 5-min incubation at room temperature. The cells were lysed with lysis buffer (R&D Systems, Minneapolis, MN, USA), and the levels of phosphorylated ERK1/2 were measured in the cell lysates using an ELISA kit (R&D Systems) for human phospho-ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187). The concentrations of phospho-ERK1/2 in the cell lysates were determined using a calibration curve with recombinant human phospho-ERK2 (Thr85/Tyr187).
7
Quantification of Inflammatory Cytokines in Brain Tissue
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Brains were collected and rinsed with PBS to remove excess blood, chopped into 1–2 mm pieces and homogenized with a tissue homogenizer. A total of 1.0 mL of Lysis Buffer (R&D Systems) was added. Brains were lysed at room temperature for 30 min with gentle agitation and centrifuged to remove debris. An aliquot of each tissue lysate was removed and assayed for levels of IL-1β and IL 18. The levels of IL-1β and IL 18 in tissues surrounding the cortical contusion site were performed by the specific ELISA kits according to the manufacturer’s instructions (R&D Systems, Inc., Minneapolis, MN, USA).
8
Quantitative Protease Array Analysis
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A Human Protease Array Kit (R&D Systems, Minneapolis, MN, USA) was used as previously reported [23 (link)]. 786-O and Caki-1 cells were treated with fisetin (0, 60 μM) for 24 h, then lysed in the lysis buffer (R&D Systems). The nitrocellulose membranes, including 34 different capture antibodies, were blocked and washed for 10 mins, then incubated with Streptavidin-HRP in the array buffer. The signal was visualized by an enhanced chemiluminescence analysis system (Fuji Film, Tokyo, Japan).
9
Western Blot Analysis of ANXA1 in HNSCC
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Proteins were extracted from HNSCC cells transfected with either pre-miR196a, pre-miR196b or a non-targeting control at 72 h post-transfection using lysis buffer (R&D Systems). Protein lysates were resolved by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred subsequently to nitrocellulose membranes (Amersham Protran, GE Healthcare).
The membranes were blocked for 1 h with Odyssey blocking buffer and incubated overnight with rabbit polyclonal ANXA1 (at 1:1,000 dilution) raised in our laboratory against the human recombinant protein and the anti-β-actin (dilution 1:10,000 for 1 h; from Sigma Aldrich # AC15). The IRDye Infrared Fluorescent secondary antibodies Goat anti-Rabbit IRDye 800CW and Goat anti-Mouse IRDye 680RD were used for detection. Membranes were scanned with the Odyssey Fc Dual-Mode Imaging System (LI-COR Biosciences) using the red (700 nm) and green (800 nm) channels.
The membranes were blocked for 1 h with Odyssey blocking buffer and incubated overnight with rabbit polyclonal ANXA1 (at 1:1,000 dilution) raised in our laboratory against the human recombinant protein and the anti-β-actin (dilution 1:10,000 for 1 h; from Sigma Aldrich # AC15). The IRDye Infrared Fluorescent secondary antibodies Goat anti-Rabbit IRDye 800CW and Goat anti-Mouse IRDye 680RD were used for detection. Membranes were scanned with the Odyssey Fc Dual-Mode Imaging System (LI-COR Biosciences) using the red (700 nm) and green (800 nm) channels.
10
Colorimetric Caspase Activity Assay
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Cells were harvested, washed with cold PBS, and incubated with a lysis buffer (R&D Systems, Inc., Minneapolis, MN, USA) for 10 min on ice. The lysed cells were centrifuged at 10,000× g for 1 min, and 100 μg of protein was added to the reaction mixture containing 2× reaction buffer and substrates of colorimetric tetrapeptides, including DEVD-pNA for caspase-3, IETD-pNA for caspase-8, and LEHD-pNA for caspase-9. The reaction mixture was incubated at 37 °C for 2 h, and then enzymatic release of p-nitroaniline was quantitated at 405 nm using a multi-wall reader (Thermo Fisher Scientific).
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