LRH-1 variant protein expression levels were measured using western blot analysis. HEK293T cells were seeded into 6-well plates and incubated at 37 °C in complete media (DMEM supplemented with 10% FBS and 1% penicillin/streptomycin) until ~70% confluent. Each well was then transiently transfected using PolyJet™ with an LRH-1 plasmid under constitutive activation via CMV promoter (1.0 μg/well). Transfection was ended after 4 hours of incubation at 37 °C via replacement of media with complete media. Cells were incubated for an additional 44 hours, trypsinized to remove from 6-well plate, washed with PBS, and frozen at −80 °C until western blot analysis. Cells were disrupted in lysis buffer (10 mM HEPES pH 7.5, 500 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS) in the presence of cOmplete Protease tablets (Roche, Indianapolis, IN), incubated on ice for 30 minutes, and centrifuged at 17,500xg for 15 minutes at 4 °C. The supernatant was quantified for total protein content via BCA assay. 5 μg total protein of each LRH-1 variant was loaded onto SDS-PAGE and was sequentially blotted for LRH-1 and β-actin.
Complete protease tablets
Manufactured by Roche
Sourced in China
Complete Protease tablets are a laboratory product designed to assist in the digestion and breakdown of proteins. They contain a mixture of enzymes that facilitate the hydrolysis of peptide bonds, enabling the efficient processing of protein samples for various analytical and experimental purposes.
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5 protocols using complete protease tablets
1
Quantitative LRH-1 Variant Protein Levels
2
Immunoprecipitation and Kinase Assay of Tagged Proteins
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PF expressing HA-tagged proteins washed in PBS containing 10 mM glucose. They were permeabilized with digitonin and the organellar pellet was extracted with sodium carbonate as described [29 (link)] except that Complete Protease tablets (Roche) were included for both digitonin permeabilization and carbonate extraction, to reduce protein degradation. Cell lysates from PF cells expressing either V5-tagged MEKK1 or HA-tagged FHK along with the untransfected parental line 29–13 were prepared as described [30 (link)]. Epitope-tagged proteins were immunoprecipitated from cell lysates prepared from 108 cells. Kinase assays were done as described [31 (link)] using 32PγATP with 5 μg myelin basic protein as an exogenous substrate. Reactions were resolved by SDS-PAGE and transferred to nitrocellulose and labeled proteins detected by phosphorimaging. Blots were subsequently probed with antibodies directed against either V5 or HA.
3
Cell Lysis and Protein Extraction
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Cells cultured on cell culture plates were washed once with 1× PBS before lysis in an appropriate volume of RIPA buffer on ice (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP-40, 1% Triton X-100, 0.1% SDS, and 0.5% Na-deoxycholate, supplemented with complete protease tablets [Roche] and phosphatase inhibitor cocktails [Sigma-Aldrich]). Cells were scratched off the plate into a reaction tube, which was vortexed every 5 min within 30 min. After ultrasonication for 10 min, lysates were centrifuged for 15 min at 21,000g at 4°C. The supernatant was mixed with 2× Laemmli buffer before SDS–PAGE and immunoblotting.
4
Fungal Burden and Cytokine Response in Murine Cryptococcosis
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Mice were infected with strain XL280 or JEC21 via intranasal instillation. At 3, 7, and 10 days post infection lung tissues were excised, homogenized in 1 ml PBS, and 50 µl were diluted and plated on YPD to determine fungal burden at designated time points, as described previously. 1 ml of 2× protease inhibitor buffer (containing PBS, Complete Protease tablets (Roche) and 0.05% Triton X100) was added and the homogenate was centrifuged at 3,500 RPM for 15 min at 4°C. Cytokine production in the pulmonary tissues was measured using the Bio-Plex Protein Array System (Luminex-based technology) (Bio-Rad Laboratories, Hercules, CA). The supernatants of the homogenized lungs were assayed for the presence of interferon (IFN)-γ, interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12 p70, IL-17, tumor necrosis factor (TNF)-α, and granulocyte-colony stimulating factor [G-CSF] levels as well as chemokines (macrophage inflammatory protein [MIP]-1α (CCL3), MIP-1β (CCL4), macrophage chemoattractant protein [MCP]-1 (CCL2), and keratinocyte-derived chemokine (KC) (CXCL1)).
5
Deacetylation Assay with Hydrolase Inhibitors
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Hlat protease inhibitor cocktail was purchased from Thermo Scientific, cOmplete protease tablets were from Roche, AEBSF, aprotinin, bestatin, and pepstatin A were obtained from Sangon Biotech Co., Ltd (Shanghai, China). The deacetylation assays were performed as described above except for the addition of a variety of hydrolase inhibitors at appropriate concentrations. The solvents of these inhibitors, that is, dimethyl sulfoxide and ethanol, were also tested as controls.
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