CD4+CD45RA+ cells were plated at 2 × 106 cells per condition for Western blotting. The cells were lysed with lysis buffer containing 2.5 mM sodium pyrophosphate, 1 mM NA3VO4, and 1 mM phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology). The cell lysates were resolved with 5–15% gradient SDS-PAGE (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% milk in TBS (20 mM Tris and 500 mM NaCl) and 0.1% Tween 20 at room temperature for 1 h followed by overnight incubation at 4°C with primary Abs against IL-1RI (Abcam), IRF4 (Santa Cruz Biotechnology), RORc (Abcam), β-actin (Sigma-Aldrich). Secondary HRP-conjugated Ab (Santa Cruz Biotechnology) was added at a dilution of 1/2000 for 1 h and the protein bands were detected with an ECL Detection System (Santa Cruz Biotechnology).
Sodium pyrophosphate
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sodium pyrophosphate is a chemical compound with the formula Na4P2O7. It is a white, crystalline powder that is soluble in water. Sodium pyrophosphate is used as a buffer, chelating agent, and emulsifier in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Phenylmethylsulfonyl fluoride, by Santa Cruz Biotechnology (1 mentions)
Ecl detection system, by Santa Cruz Biotechnology (1 mentions)
Na3vo4, by Santa Cruz Biotechnology (1 mentions)
Il 1ri, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Sodium orthovanadate, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Santa Cruz Biotechnology (1 mentions)
Cell culture reagents, by Lonza (1 mentions)
Celltiter blue cell viability assay, by Promega (1 mentions)
Phenylmethanesulfonyl fluoride pmsf, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer system, by Santa Cruz Biotechnology (1 mentions)
Phosphatase inhibitor cocktail c, by Santa Cruz Biotechnology (1 mentions)
2 protocols using sodium pyrophosphate
1
Western Blot Analysis of T Cell Subsets
2
Molecular Profiling of SIRT1 and AMPK
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Cell culture reagents were purchased from Lonza (Basel, Switzerland) and Gibco-BRL (Grand Island, NY). General laboratory chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Carlo Erba (Milano, Italy).
Ripa Lysis Buffer System, Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail C, phenyl-methane-sulfonyl-fluoride (PMSF), Sodium Orthovanadate and Sodium Pyrophosphate were obtained from Santa Cruz Biotechnology (California, USA). CellTiter-Blue Cell Viability Assay was purchased from Promega (Madison, USA).
Reagents, protein markers and membrane for Western Blot were from Biorad Laboratories (California, USA), Rabbit polyclonal antibody against SIRT1 (H-300, sc-15404), mouse monoclonal antibody against Actin (C-2, sc-8432), goat anti-rabbit IgG-HRP antibody (sc-2004) and goat anti-mouse IgG-HRP antibody (sc-2005) were purchased from Santa Cruz Biotechnology (California, USA).
Rabbit polyclonal antibody against Phosho-AMPKα (Thr172, #2531), rabbit polyclonal antibody against Total AMPKα (#2532) were from Cell Signaling Technologies (Massachussets, USA). Luminata Crescendo Western HRP Substrate for chemiluminescent detection of bands were from Millipore (Massachussets, USA).
Ripa Lysis Buffer System, Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail C, phenyl-methane-sulfonyl-fluoride (PMSF), Sodium Orthovanadate and Sodium Pyrophosphate were obtained from Santa Cruz Biotechnology (California, USA). CellTiter-Blue Cell Viability Assay was purchased from Promega (Madison, USA).
Reagents, protein markers and membrane for Western Blot were from Biorad Laboratories (California, USA), Rabbit polyclonal antibody against SIRT1 (H-300, sc-15404), mouse monoclonal antibody against Actin (C-2, sc-8432), goat anti-rabbit IgG-HRP antibody (sc-2004) and goat anti-mouse IgG-HRP antibody (sc-2005) were purchased from Santa Cruz Biotechnology (California, USA).
Rabbit polyclonal antibody against Phosho-AMPKα (Thr172, #2531), rabbit polyclonal antibody against Total AMPKα (#2532) were from Cell Signaling Technologies (Massachussets, USA). Luminata Crescendo Western HRP Substrate for chemiluminescent detection of bands were from Millipore (Massachussets, USA).
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