Hela cells were lysated after 48 h transfection with pRRL-PGK-T2AGFP–STAT5B WT and mutant constructs. Non-transfected cell line and cell transfected with an empty vector were used as internal control for the reaction. Standard western blot analysis was carried out. RIPA buffer supplemented with protease inhibitors cocktail (Sigma Aldrich) and phosphates inhibitor cocktail (Sigma Aldrich) was used for protein extraction. Proteins were transferred to a polyvinylidene difluoride membrane, after which the membrane was blocked with Super Block (Thermo Fisher) blocking buffer for 1 h. Primary antibodies for STAT5B were obtained from Cell Signal Technology: STAT5B Rabbit mAb#9363 dilution 1:1,000 in 5% milk and Phospho-Stat5 (Tyr694) (D47E7) Rabbit mAb #4322 dilution 1:500 in Super Block. Monoclonal Anti-Actin (Clone AC-40) and anti-GFP (clone GFP-20) antibodies were purchased from Sigma Aldrich and used to a working dilution of 1:1,000. Membranes were incubated overnight at +4 °C. Uncropped representative WB images are shown in Supplementary Fig. 11 .
Phosphates inhibitor cocktail
Manufactured by Merck Group
The Phosphates inhibitor cocktail is a laboratory reagent designed to inhibit the activity of phosphates in samples. It is used to prevent the degradation of phosphorylated biomolecules during sample processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitors cocktail, by Merck Group (1 mentions)
Phospho stat5 tyr694 d47e7, by Cell Signaling Technology (1 mentions)
Superblock, by Cell Signaling Technology (1 mentions)
Superblock, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated hrp anti rabbit antibodies, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti human β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
2 protocols using phosphates inhibitor cocktail
1
Analysis of STAT5B Phosphorylation in HeLa Cells
2
FOXO3 Expression in NSLCL Cells Treated with ESMC
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NSLCL cells treated with ESMC for 48 h were lysed with RIPA lysis buffer containing protease inhibitor cocktail and phosphates inhibitor cocktail (Sigma-Aldrich; Merck KGaA) on ice for 30 min, then lysis buffer was collected, and centrifuged at 12,000 g, 4°C for 10 min. The protein lysates were resolved by SDS-PAGE, and separated proteins were transferred to PVDF membranes and blocked with 5% skimmed milk for 2 h. The primary antibodies used for western blotting were rabbit anti-human FOXO3 antibody (1:1,000; Santa Cruz Biotechnology, Inc.) and rabbit anti-human β-actin antibody (1:1,000; Santa Cruz Biotechnology, Inc.). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (1:5,000; Santa Cruz Biotechnology, Inc.) were used as the secondary antibodies. The blots were incubated with the respective antibodies overnight at 4°C under gently shaking. Finally, the proteins were detected by using horseradish peroxidase labeled secondary antibodies and an enhanced chemiluminescence detection system.
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