Cells were lysed in lysis buffer (20 mM Tris-HCl [pH 7.4], 5 mM EDTA [pH 8.0], 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 1% NP-40) containing aprotinin, leupeptin, PMSF, and phosphatase inhibitors cocktail 3 (Sigma). Protein samples were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with primary antibody overnight at 4˚C. Primary antibodies against the following proteins were obtained from the indicated suppliers: C/EBPβ, Sestrin2, phospho-p70S6K (T389), p70S6K, p-Rb (S807/811) were from Cell Signaling Technology; Rb was from Santa Cruz Biotechnology; and β-actin (1:5,000) from Sigma.-Membranes were washed three times with TBST and incubated with HRP-conjugated anti-mouse (Santa Cruz Biotechnology) or anti-rabbit secondary antibody (Cell Signaling Technology). HRP was detected using the ECL reagent (BioNote).
Phosphatase inhibitors cocktail 3
Manufactured by Merck Group
Phosphatase inhibitors cocktail 3 is a laboratory reagent used to inhibit the activity of phosphatases, which are enzymes that remove phosphate groups from proteins. This product contains a mixture of various phosphatase inhibitors, allowing for the preservation of the phosphorylation state of proteins in biological samples during analysis.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Roche (2 mentions)
Anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
P rb s807 811, by Cell Signaling Technology (1 mentions)
Sestrin2, by Cell Signaling Technology (1 mentions)
C ebpβ, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Furimazine, by Promega (1 mentions)
Eos 1100d camera, by Canon (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Foxo3a, by Abcam (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
6 protocols using phosphatase inhibitors cocktail 3
1
Immunoblotting Protocol for Protein Analysis
2
Identification of TRIM9 Interactome
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HEK293T cells stable expressing V5-TRIM9 were collected and lysed with NP40 buffer (50 mM HEPES, pH 7.4, 150mM NaCl, 1mM EDTA, 1% (v/v) NP40) supplemented with a complete protease inhibitor cocktail (Roche) and phosphatase inhibitors cocktail 3 (Sigma). Post-centrifuged supernatants were pre-cleared with protein A/G beads at 4°C for 2 hours and mixed with mouse anti-V5 and protein A/G beads for 4 hours at 4°C. Precipitates were washed extensively with lysis buffer and separated by SDS-PAGE. After silver staining, specific protein bands were excised and analyzed by ion-trap mass spectrometry at the Harvard Taplin Biological Mass Spectrometry facility, and amino acid sequences were determined by tandem mass spectrometry and database searches.
3
Rapamycin and Torin 1 Protocol
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Rapamycin and Torin 1 were purchased from Calbiochem (Merck) and resuspended in DMSO. Phosphatase inhibitors cocktail 3 (P0044) is from SIGMA. Furimazine was purchased from Promega (France).
4
Identification of TRIM9 Interactome
5
Quantification of FOXO3A and AKT1 Phosphorylation
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Ovaries and rat tongue were homogenized using cryogrinding and lysed in RIPA buffer with 1% v/v protease inhibitor cocktail and 1% v/v phosphatase inhibitors cocktail 3 (Sigma-Aldrich). Protein concentration was determined using the Bio-Rad DC Protein assay (Bio-Rad). Total protein of 40 mg for PN day 8 ovaries and tongue, and 13 mg for PN day 6 ovaries were loaded on a Bis-Tris 4-12% Gel and electrophoretic separated. The proteins were transferred to an Invitrolon PVDF membrane (Life Technologies) and equal protein load was checked using CPTS stain (Bickar & Reid 1992) followed by destaining in a 5% w/v BSA (Sigma-Aldrich) solution in Tris Buffed Saline and drying of the membrane after an ethanol wash. Western blotting was carried out as described previously (Naryzhny 2009 (link)) using rabbit antibodies raised against FOXO3A (AbCam, Cambridge, UK), phospho-FOXO3A (AbCam), phospho-AKT1 S473 (AbCam) and peroxidase labelled donkey anti-rabbit secondary antibody (Agrisera, Va ¨nnas, Sweden). The blot was developed with SuperSignal West Femto Substrate (Thermo Fisher Scientific, Hvidovre, Denmark) and imaged with a Canon EOS 1100D camera (Canon, Tokyo, Japan) cooled with dry ice.
6
Drosophila Ovary Protein Extraction and Immunofluorescence
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To prepare ovary protein extracts, Drosophila ovaries were disrupted through sonication in Lysis Buffer (150 mM KCl, 75 mM HEPES, pH 7.5, 1.5 mM EGTA, 1.5 mM MgCl 2 , 15% glycerol, 0.1% NP-40) containing 1x protease inhibitors cocktail (Roche) and 1x phosphatase inhibitors cocktail 3 (Sigma). For protein extracts from S2 cells, harvested cells were washed with PBS 1x supplemented with protease inhibitors cocktail (Roche). Lysis buffer was then added and cells were disrupted by sonication. Samples were then centrifuged and supernatant was collected for analyses.
Immunofluorescence and F-actin staining Drosophila ovaries were fixed using a 4% paraformaldehyde solution (in PBS), followed by washing steps with 0.05% PBT (PBS with 0.05% TWEENâ 20 (Sigma)), 1 hour-blocking with 10% BSA in 0.2% PBT, and overnight incubation at room temperature with primary antibodies diluted in 0.05% PBT (supplemented with 1% BSA). Ovaries were then washed with 0.05% PBT containing 1% BSA, incubated 2 hours with the secondary antibody and washed with 0.05% PBT before mounting in Vectashield with DAPI. The primary antibodies used are indicated in the key resources table. F-actin staining was performed by adding Phalloidin-TRITC at 1 mg/mL to the paraformaldehyde solution during the fixation.
Immunofluorescence and F-actin staining Drosophila ovaries were fixed using a 4% paraformaldehyde solution (in PBS), followed by washing steps with 0.05% PBT (PBS with 0.05% TWEENâ 20 (Sigma)), 1 hour-blocking with 10% BSA in 0.2% PBT, and overnight incubation at room temperature with primary antibodies diluted in 0.05% PBT (supplemented with 1% BSA). Ovaries were then washed with 0.05% PBT containing 1% BSA, incubated 2 hours with the secondary antibody and washed with 0.05% PBT before mounting in Vectashield with DAPI. The primary antibodies used are indicated in the key resources table. F-actin staining was performed by adding Phalloidin-TRITC at 1 mg/mL to the paraformaldehyde solution during the fixation.
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