For total protein extraction, hPSCs were lysed with RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche) and phosphatase inhibitors cocktail 2 and 3 (Sigma-Aldrich). For histone extraction we used the Histone extraction protocol for western blot from Abcam (Cambridge; UK) web page. Cell lysates were separated by molecular weight using SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. Protein was detected using the Odyssey Infrared Imaging System (Li-cor Biosciences; Lincoln, NE, USA). To detect KDM5A and NUP98-KDM5A was used the α-KDM5A antibody (ab70892, Abcam). Also used α-NUP98 (ab50610, Abcam), α-HIF1A (610959, BD Bioscience; San Jose, CA, USA), α-γ-H2AX (#9718, Cell signalling; Danvers, MA, USA). An α-β-Actin (A5441, Sigma-Aldrich) was used as a loading control for total protein extractions and α-H3 (ab1791, Abcam) was used as a loading control in histone extractions. Western blotting was carried out using standard procedures. Quantification of band intensity and normalization was carried out using ImageJ (NHI, Bethesda, Maryland, USA, https://imagej.nih.gov/ij ).
Phosphatase inhibitors cocktail 2 and 3
Manufactured by Merck Group
Phosphatase inhibitor cocktails 2 and 3 are laboratory reagents designed to inhibit the activity of phosphatases, a class of enzymes that remove phosphate groups from proteins. These cocktails are used to preserve the phosphorylation state of proteins in biological samples, which is important for studying cellular signaling pathways and protein function.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (3 mentions)
β actin, by Merck Group (3 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Protease inhibitors cocktail, by Merck Group (2 mentions)
Trans blot turbo, by Bio-Rad (2 mentions)
Complete protease inhibitor cocktail, by Merck Group (2 mentions)
Ab70892, by Abcam (1 mentions)
Ab50610, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
α γh2ax, by Cell Signaling Technology (1 mentions)
α β actin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Hybond ecl, by GE Healthcare (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Nitrocellulose, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Bcl xl, by Santa Cruz Biotechnology (1 mentions)
Rad50, by Cell Signaling Technology (1 mentions)
Mre11a, by Cell Signaling Technology (1 mentions)
6 protocols using phosphatase inhibitors cocktail 2 and 3
1
Histone and Total Protein Extraction Protocols
2
Pyocyanin Cytotoxicity Assay in MEFs and Fibroblasts
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MEF Ttc19+/− and MEF Ttc19−/−, as well as human fibroblasts from healthy donor or patients, were seeded in standard 6-well plates at a density of 7 × 104 cells/well for MEFs or in 100 mm dishes at a density of 2.2 × 105 cells/dish for fibroblasts, and allowed to attach overnight in complete DMEM medium. From the following day, cells were incubated with 1.5 or 0.8 µM of PYO (by changing the medium with freshly prepared one containing PYO), for MEFs and fibroblasts, respectively, or the corresponding amount of DMSO (vehicle). Incubation was carried out for 24, 48, or 72 h in standard DMEM with 2% of FBS. At the end of the treatment, the medium was removed and 150 µl of Lysis Buffer (25 mM Tris pH 7.8 + 2.5 mM EDTA + 10% glycerol + 1% NP40 + 2 mM dithiothreitol (DTT) + 1% phosphatase inhibitors cocktail 2 and 3 (Sigma-Aldrich) + 1% protease inhibitors cocktail (Sigma-Aldrich)) were added to each well. The plates were then frozen at −80 °C. Total extracts were collected from each well after scraping, vortexed for 10 s, and then centrifuged at 20,000 × g for 10 min at 4 °C. To enhance protein separation, samples were solubilized for 30 min at room temperature (RT) in Sample Buffer (10% glycerol + 42 mM Tris/HCl pH 6.8 + 3% sodium dodecyl sulfate + 33 mM DTT + bromophenol blue).
3
Hydroxytyrosol Dose-Dependent Effects on Protein Expression
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Semiconfluent A374 cells were treated with 250 μM, 375 μM, and 500 μM of hydroxytyrosol and HT-144 cells with 250 μM, 350 μM, and 450 μM of hydroxytyrosol for 24 h and 48 h; therefore, the cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS) containing proteases and phosphatase inhibitors (4 mM PMSF and protease inhibitors cocktail, phosphatase inhibitors cocktail 2 and 3, Sigma). The protein concentration of cellular lysates was determined by Bradford protein assay (Bio-Rad laboratories GmbH, München, Germany). Furthermore, 20 μg or 40 μg of total cell extracts were resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and absorbed to nitrocellulose membrane (Hybond ECL, GE Healthcare, Biosciences, Pittsburgh, PA, USA) to perform western blot experiments as previously reported [49 (link)]. The membranes were processed and the protein bands were scanned and quantified by densitometric analysis, using Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). The results of quantitative analysis are reported in the histograms as mean values of several western blot experiments and are expressed as a percent of unstimulated cells, normalized for actin amount.
4
Protein Extraction and Immunoblotting Protocol
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The cell pellets were lysed with 1 % NP-40 lysis buffer (20 mmol/L Tris–HCl, pH 7.5; 1 mmol/L EDTA; 150 mmol/L NaCl; 1 % NP-40) phosphatase inhibitors cocktail 2 and 3 (Sigma) and containing protease inhibitors (Roche) for 30 to 45 min at 4 °C. Protein lysates were prepared after calculating protein concentration using the Bradford reagent (Biorad) and 50 μg of protein was resolved on 10 % SDS-PAGE followed by transferring to nitrocellulose (Millipore) [30 (link)]. Immunoblotting was performed with primary antibodies against BCL-xL (Santa Cruz Biotechnology) and β-actin (Sigma). The secondary anti-mouse antibodies were purchased from Thermo-Fisher Scientific.
5
Quantitative Western Blot Analysis
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After treatment, cells were washed in PBS and lysed in RIPA buffer (Sigma) supplemented with Complete Protease inhibitor cocktail (ROCHE) and phosphatase inhibitors cocktail 2 and 3 (Sigma). Lysed cells were harvested by scraping, and proteins were analyzed by western blot. Equivalent amounts of protein were loaded on a 4–12% gradient SDS-polyacrylamide gel (BioRAD) and transferred for 30 min in a TransBlot turbo (BioRAD) onto nitrocellulose membranes. Membranes were blocked in 5% milk or 3% bovine serum albumin (BSA) and incubated with antibodies as indicated: MRE11a (Cell Signaling, 4847, 1:1000); RAD50 (Cell Signaling, 3427, 1:1000); NBS1 (Cell Signaling, 14956, 1:500); p21 (Cell Signaling, 2947, 1:1000); and pRb (Cell Signaling, 9301, 1:500). β-actin (Sigma, A5316, 1:10,000 1 h RT) was used as a housekeeping control for the total levels of protein loaded. Membranes were washed in Tris-buffered saline (TBS) with Tween 20 and incubated with secondary antibodies from Licor Biosciences. Licor antibodies used were Goat anti-Mouse 925–68020 (1:15 000) and Goat anti-Rabbit 925–32211 (1:15 000). Blots were scanned on the Licor Odyssey scanner according to the manufacturer’s instructions.
6
Western Blot Analysis of SMPD1 and Gasdermin D
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After treatment, cells were washed in phosphate-buffered saline (PBS) and lysed in RIPA buffer (Sigma) supplemented with Complete Protease inhibitor cocktail (ROCHE) and Phosphatase inhibitors cocktail 2 and 3 (Sigma). Lysed cells were harvested by scraping, and proteins were analyzed by Western blot. Equivalent amounts of protein were loaded on a 4–12% gradient SDS-polyacrylamide gel (BioRAD) and transferred for 30 min in a TransBlot turbo (BioRAD) onto Nitrocellulose membranes. Membranes were blocked in 5% milk or 3% BSA and incubated with antibodies as indicated: Anti-SMPD1 (OTI3H7, NBP2-45889, Novus Biologicals, or Clone # 563418, R&D Systems 1:1000, overnight, 4 °C), Gasdermin D (96458, Cell Signaling, 1:1000, overnight, 4 °C), GAPDH (D16H11, Cell Signaling, 1:5,000, 1 h RT), β-actin (Sigma, A5316, 1: 5,000 1 h RT) was used as a housekeeping control for the total levels of protein loaded. Membranes were washed in TBST and incubated with secondary antibodies from Licor Biosciences. Licor antibodies used were goat anti mouse 925–68020 (1:15,000) and goat anti rabbit 925–32211 (1:15,000). Blots were scanned on the Licor Odyssey scanner or Azure scanner according to manufacturer’s instructions. Bands were quantified manually using ImageJ to calculate the Integrated Density values of the band of interest and normalizing it to the loading control in the same lane.
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