Harvested cultured cells were lysed in lysis buffer (Cell Signaling Technology, Danvers, MA) containing phosphatase inhibitor cocktail C (Santa Cruz, Dallas, TX). The concentration of proteins in cell lysates was quantified by the Enhanced BCA Protein Assay Kit (Pierce Biotechnology, Inc., Waltham, MA, USA) and 25 μg of proteins was loaded in each lane. Western blotting was performed as previously described52 (link). Primary antibodies were incubated for overnight at 4 °C: anti-KIF3A (1:2000; Abcam, Cambridge, MA), β-catenin (1:1000; Cell Signaling Technology), DVL-2 (1:1000; Cell Signaling Technology), β-arrestin (1:1000; BioLegend Inc., San Diego, CA), axin (1:1000; Cell Signaling Technology), LRP (1:1000; Cell Signaling Technology), phosphorylated LRP (1:1000; Cell Signaling Technology) and β-actin (1:2000; Santa Cruz). Incubation with HRP-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies (1:1000; Enzo Life Sciences, Inc., Farmingdale, NY) antibodies was performed for 1 hr at room temperature. The full blots of the cropped images are presented in Supplementary Figures 4 and 5 .
Phosphatase inhibitor cocktail c
Manufactured by Santa Cruz Biotechnology
Sourced in Italy
Phosphatase inhibitor cocktail C is a solution containing a combination of specific phosphatase inhibitors. It is designed to inhibit a wide range of protein phosphatases, including serine/threonine and tyrosine phosphatases, in order to preserve the phosphorylation state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lysis buffer, by Cell Signaling Technology (2 mentions)
Enhanced bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti kif3a, by Abcam (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Protein g sepharose, by GE Healthcare (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)
Sodium pyrophosphate, by Santa Cruz Biotechnology (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)
3 protocols using phosphatase inhibitor cocktail c
1
Western Blot Analysis of Wnt Pathway Proteins
2
Immunoprecipitation of β-arrestin and Axin
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For immunoprecipitation reactions, cells were lysed in lysis buffer (Cell Signaling Technology) containing phosphatase inhibitor cocktail C (Santa Cruz). The concentration of proteins in cell lysates was quantified by the Enhanced BCA Protein Assay Kit (Pierce Biotechnology, Inc.). Each 5 ul antibody for β-arrestin (1:200, BioLegend Inc.) or axin (1:200, Cell Signaling Technology) per 1 mg lysate was incubated overnight at 4 °C with constant rotation. The next day, a protein G sepharose (GE Healthcare Bio-Sciences Corp.) bead was added to each lysate for 4 h at 4 °C with constant rotation. Unbound proteins were removed by four washes using lysis buffer and proteins were eluted from the beads by the addition of 40 μl sample buffer. SDS-PAGE was used to analyze 20 μl samples.
3
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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