In this study, cells were incubated in RIPA buffer (#P0013B; Beyotime, Shanghai, China) containing protease inhibitor (#K1007; APExBIO, Houston, America) and phosphatase inhibitor (#K1015; APExBIO) at 4°C. After 30 min, lysates were centrifuged at 12,000 rpm for 15 min at 4°C to obtain the supernatant. Proteins were quantified with a BCA assay kit (#P0010S, Beyotime), and 20 μg per lane were separated by SDS-PAGE and transferred to PVDF membranes (#IPVH00010, Millipore, Shanghai, America). Then, the membrane was incubated for 1 h at room temperature with HRP-conjugated antibody and developed using ECL reagent (#P0018S, Beyotime). Furthermore, antibodies against flag Tag (#8146, 1:1000), phospho-AKT (Ser473) (#13038, 1:1000), AKT (#4691, 1:3000), Bcl-2 (#15071, 1:1000), BAX (#5023, 1:1000) and cleaved Caspase-3 (#9661, 1:3000) were ordered from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA). Besides, cleaved PARP-1 (#ab32064, 1:1000) was purchased from Abcam (Abcam, Cambridge, UK), antibodies against β-actin (#YT0099, 1:3000) and HRP Goat anti-mouse IgG(H + L) (#RS0001,1:5000) from ImmunoWay (Jiangsu, China) and anti-rabbit IgG HRP-linked antibody (#7074, 1:5000) from Cell Signaling Technology.
Phosphatase inhibitor
Manufactured by Apexbio
Sourced in United States
Phosphatase inhibitor is a lab equipment product that functions to inhibit the activity of phosphatase enzymes. Phosphatases are responsible for the removal of phosphate groups from various biomolecules, and their inhibition can be important in certain experimental and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Apexbio (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Cleaved parp1, by Abcam (1 mentions)
Ab219800, by Abcam (1 mentions)
Af7021, by Affinity Biosciences (1 mentions)
Sds page sample buffer, by GenStar (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Ag 20b 0042 c100, by Adipogen (1 mentions)
Protease inhibitor cocktail, by Apexbio (1 mentions)
Immobilon western chemilum hrp substrate, by Merck Group (1 mentions)
Horse radish peroxidase conjugated secondary antibodies, by Signalway Antibody (1 mentions)
6 well plate, by Corning (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
4 protocols using phosphatase inhibitor
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Apoptosis Regulators
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Ovaries fragments or cells were lysed in RIPA buffer with protease inhibitor (Apexbio) and phosphatase inhibitor (Apexbio) for 10 mins and centrifuged at 12,000 g for 10 mins at 4 °C. The supernatant mixed with SDS‐PAGE sample buffer (GenStar) was denatured at 100 °C for 10 mins. Denatured protein was separated on 10–15% SDS‐PAGE gel and transferred to Polyvinylidene Fluoride membrane. Blocked with 5% non‐fat dry milk, the membranes were incubated with primary antibodies of GSDMD (Abcam, ab219800), IL1beta (Abcam, ab205924), caspase 1 (AdipoGen, AG‐20B‐0042‐C100), caspase 11 (AdipoGen, AG‐20B‐0060‐C100) and GAPDH (Affinity, AF7021) overnight at 4 °C and the corresponding secondary antibodies for 1 h at room temperature. The blots were imaged with ChemiDoc imaging system (Bio‐Rad).
3
Western Blot Analysis of Protein Expression
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We washed the cells cultured in 6-well plates (Corning Life Sciences, USA) three times in PBS and lysed them on ice in RIPA buffer, protease-inhibitor cocktail, and phosphatase-inhibitor (APExBIO, USA) solution. They were then mechanically broken down using a syringe. The suspension was centrifuged (4 ℃) and the supernatant was collected. An enhanced BCA protein assay kit (Beyotime Biotechnology, China) was used to determine the protein concentration of the supernatant. Proteins (50 μg) were separated on 10% SDS–polyacrylamide gels and transferred onto PVDF membranes (Millipore, USA). The PVDF membranes were blocked with 5% skim milk in TBS, then incubated with primary antibodies (overnight, 4 °C). After three washings in TBS-T, the membranes were incubated (25 °C, 1 h) with horse radish peroxidase-conjugated secondary antibodies (1:3000; Signalway Antibody, USA) and exposed to immobilon western chemilum hrp substrate (Millipore, USA). To confirm equal protein loading, we used anti-β-actin antibodies (1:2000, Signalway antibody, USA) to re-probe. The antibodies used are listed in Additional file 1 : Table S2.
4
Metabolite-Induced Protein Responses
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Cells were washed with cold phosphate buffered saline (PBS) for three times, scraped and centrifuged at 1,000 rpm for 5 min. The cell pellets were resuspended in mammalian protein extraction reagent (M-PER, Pierce/Thermo Fisher Scientific, cat. no. 78503) containing protease inhibitor (ApexBio Technology, Houston, TX, USA, cat. no. K1007) and phosphatase inhibitor (ApexBio Technology, cat. no. K1013), and lysed on ice for 30 min. The supernatants were collected by centrifugation at 18,000 g for 10 min. Protein concentration was determined by bicinchoninic acid (BCA) Protein Assay (Beyotime Biotechnology, Beijing, China, cat. no. P0011). The cell lysates were then diluted with M-PER lysis to 3 μg/μL and incubated with given metabolites or solvents for 1 hr at 25 ˚C. Specifically, the glycolytic metabolites were administered at dosages detailed as follows: D-Fructose 1,6-bisphosphate (FBP, 200 μM), D-fructose 6-phosphate (F6P, 100 μM), D-glucose 6phosphate (G6P, 100 μM), D-ribose 5-phosphate (R5P, 30 μM), D/L-glyceraldehyde-3-phosphate (G3P, 30 μM), D-2-phosphoglycerate (2PG, 10 μM) from Toronto Research Chemicals, North York, Ontario, Canada. D-3phosphoglycerate (3PG, 10 μM), phosphoenolpyruvate (PEP, 5 μM), pyruvate (Pyr, 300 μM), L-lactate (Lac, 2 mM). The dosages were set based on the intracellular concentrations of these metabolites as reported in literature 42, 43 .
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