Cells were lysed on ice with CelLytic MT reagent (Sigma) supplemented with protease and phosphatase inhibitors. Primary antibodies: TSC1 (#6935), TSC2 (#4308), phospho-S6 (#4858), S6 (#2217), p21 (#2947), Lamin B1 (#12586), LC3 A/B (#12741), phospho-ATG14-S29 (#92340), ATG14 (#96752), phospho-CREB-S133 (#9198), CREB (#9197), phospho-CHK1-S345 (#2348), phospho-WEE1-S642 (#4910), WEE1(#13084), PLK1 (#4513), γH2A.X (#9718), phospho-S6K-S371 (#9208), S6K (#2708), phospho-4EBP1-S65 (#9451), 4EBP1 (#9644), phospho-AKT-S473 (#4060), AKT (#4691), p53 (#2527), phospho-CDC2-T15 (#9111), CDC2 (#9116), and phospho-ATR-S428 (#2853) from Cell signaling (all used at a 1: 1000 dilution); β-tubulin (#10094-1-ap, 1: 4000), β-actin (#20536-1-ap, 1: 4000), and GAPDH (#10494-1-ap, 1: 10000) from ProteinTech; ATR (#A300-137A-T, 1: 1000) from Bethyl Lab, and CHK1 (#sc-8408, 1:1000) and Cyclin B1(#sc-245, 1: 1000) from Santa Cruz.
Cellytic mt reagent
Manufactured by Merck Group
Sourced in United States
CelLytic MT is a reagent designed for the efficient lysis of mammalian cells. It is a mild, non-ionic detergent-based solution that facilitates the extraction of proteins from cells while preserving their functionality and native structures.
Automatically generated - may contain errors
Lab products found in correlation
Phospho chk1 s345, by Cell Signaling Technology (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
β tubulin, by Proteintech (1 mentions)
Phospho akt s473, by Cell Signaling Technology (1 mentions)
Phospho crebs133, by Cell Signaling Technology (1 mentions)
Cyclin b1, by Santa Cruz Biotechnology (1 mentions)
Phospho atg14 s29, by Cell Signaling Technology (1 mentions)
Phospho wee1 s642, by Cell Signaling Technology (1 mentions)
Phospho 4e bp1 s65, by Cell Signaling Technology (1 mentions)
Phospho atr s428, by Cell Signaling Technology (1 mentions)
Lamin b1, by Cell Signaling Technology (1 mentions)
Phospho s6, by Cell Signaling Technology (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
Gapdh, by Proteintech (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Sds page chamber, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
8 protocols using cellytic mt reagent
1
Comprehensive Western Blot Analysis
2
Liver Protein Expression Analysis
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Livers were homogenized in CelLytic MT reagent (Sigma-Aldrich/MilliporeSigma) with protease inhibitors (Calbiochem/MilliporeSigma, Burlington, MA, USA) and subject to Western Blot analysis. Ten μg of total protein was loaded into 4–20% TGX gel in an SDS-PAGE chamber (BioRad, Hercules, CA, USA), electrophoresed, then transferred overnight to a PVDF membrane in Tris-glycine buffer containing 9% methanol. Quantification of protein expression was carried out in an Odyssey CLx Imaging System (Li-Cor Biosciences, Lincoln, NE, USA).
3
Western Blot Analysis of eNOS Phosphorylation
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After HUVEC treatments, cells (1 × 106) were lysed with CelLytic MT Reagent (Sigma-Aldrich, USA) in the presence of Protease Inhibitor Cocktail (Sigma-Aldrich, USA). The proteins were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on nitrocellulose membranes (Whatman, GE Healthcare, UK), 1 h at 100 mA. After 2 h blocking (PBS with 0.1% Tween 20 and 3% BSA), the membranes were incubated overnight at 4°C with primary mouse monoclonal anti-phospho-Ser1177 and anti-phospho-Thr495 antibodies (BD Transduction Laboratories, USA). Thereafter, the membranes were stripped and reprobed for monoclonal purified mouse anti-eNOS antibodies (BD Transduction Laboratories, USA); α-tubulin was used as the reference. The enhanced chemiluminescence (ECL) Horseradish Peroxidase (HRP) anti-mouse secondary antibody (Jackson, Baltimore, PA, USA) was incubated 1 h at 25°C, and chemiluminescence was determined (Amersham, GE Healthcare, UK). Densitometric analysis was carried out by the ChemiDoc™ MP Image Analysis Software (Bio-Rad, USA).
4
Protein Quantification in Cell Lysates
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Cells were lysed with CelLyticMT reagent (Sigma-Aldrich Chemie, Saint Quentin-Fallavier France) according to manufacturer’s recommendations. For each condition, the quantity of protein was determined by Pierce kit BCA (Thermo Fisher Scientific, Illkirch-Graffenstaden, France).
5
Western Blot Analysis of TNF-R1 and F4/80 in IECs
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Isolated IECs were lysed in CelLytic MT reagent (Sigma) before centrifugation at 10 000 rpm for 10 min to pellet cellular debris. Supernatants were mixed with 2 × Laemmli sample buffer before being separated by sodium dodecyl sulfate (SDS)–PAGE with 3–14% acrylamide gel and transferred to Hybond-P PVDF membrane (GE Healthcare, Buckinghamshire, UK) and blocking with 5% Marvel milk in with tris(hydroxymethyl)aminomethane (Tris). (Tris)-buffered saline containing Tween 20 (TTBS) immunostaining was performed with 1/1000 anti-TNF-R1 antibody (Abcam) and 1/5000 goat anti-Rabbit IgG HRP conjugate (Millipore) on a reduced gel. Macrophage expression was analysed similarly using antibody against F4/80 antigen (Abcam) at 1 : 1000 and goat anti-rat IgG-HRP (SantaCruz, at 1 : 3000), on a non-reduced gel. Washes were in TTBS. For detection, Immobilon Western chemiluminescent HRP substrate (Millipore) was applied to the membrane as recommended by the manufacturer and signal was detected, using a FluorChem E imaging system (Protein Simple). Band densities were quantified using Fiji [27 (link)].
6
Gastric IL-17 Quantification Protocol
7
Embryonic Brain Protein Extraction and Analysis
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The appropriate amount of CelLytic MT reagents (Sigma-Aldrich, St. Louis, MO, USA) (20 ml of reagent for 1 g of tissue) was added to embryonic brain tissues (3–4 embryonic brain tissues as one samples) respectively for protein extraction. The samples were then transferred (with lysis/extraction reagent) to a pre-chilled microhomogenizer and centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was isolated and maintained at −80 °C. Protein levels were determined by Nanodrop 2000 Spectrophotometer at 280 nm (Thermo Scientific, Waltham, MA, USA). Proteins were separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After the SDS/PAGE, samples were placed in 5 % skim milk at room temperature for 1 h. The phosphohistone H3 (PH3) (Ser10) antibody (Cell Signaling, 1: 1000) and the cleaved Caspase-3 (Asp175) antibody (Cell Signaling, Boston, MA, USA; 1: 500) were added to the membranes overnight at 4 °C. After that, anti-rabbit secondary antibodies (1: 1000) were added at room temperature for another 2 h. Detection was performed using ECL reagent. Results were analyzed with a gel image processing system.
8
INPP5E Expression Analysis in NTD Mice
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The expression levels of the INPP5E gene at different time points were determined by Western blot analysis. Briefly, the extracted neural tissues from control and NTD mouse model embryos at GD 11.5, GD 13.5 and GD 15.5, respectively, were homogenized in CelLytic MT reagents (Sigma-Aldrich, St. Louis, MO, USA). The extracted protein levels were determined using the Bradford spectroscopic protein assay method. Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Scientific, #88585), which was then sealed by 10% dried skimmed milk powder solution at room temperature for 1.5 h. The PVDF membranes were incubated overnight at 4°C, with the primary antibody to the INPP5E protein (1: 1000) (Santa Cruz Biotechnology), then incubated with the anti-mouse secondary antibody (1: 2000) (Santa Cruz Biotechnology) at room temperature for 2 h. Detection was performed with enhanced chemiluminescent (ECL) substrate (Thermo SuperSignalTM West Pico), and the results were analyzed using a gel image processing system. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.
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