Measuring p66Shc Signaling Pathway Activation and Mitochondrial Translocation in Cultured Cells

Mariusz R. Wieckowski1, Cláudia M. Deus2, Renata Couto2, Monika Oparka1, Magdalena Lebiedzińska‐Arciszewska1, Jerzy Duszyński1, Paulo J. Oliveira2

1 Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, 2 CNC—Center for Neuroscience and Cell Biology, University of Coimbra, Cantanhede
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 25.6
DOI:  10.1002/0471140856.tx2506s66
Online Posting Date:  November, 2015
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The adaptor protein p66Shc links membrane receptors to intracellular signaling pathways, with downstream consequences on mitochondrial metabolism and reactive oxygen species production. Moreover, p66Shc has also been implicated in cancer development, progression, and metastasis. Increased phosphorylation of serine 36 residue of p66Shc very often correlates with oxidative stress–associated pathologies. The pro‐oxidative role of p66Shc also appears to be involved in chemical toxicity, being an important component of stress responses triggered by xenobiotics. Here, we present a protocol that can be used: (a) for isolation of mitochondrial, cytosolic, and mitochondrial‐associated membrane fractions from adherent cells lines; (b) to perform p66Shc detection with specific antibodies in order to monitor its translocation between different cellular compartments in response to the oxidative stress; and (c) to modulate the p66Shc pathway with the use of pharmacological approaches or gene‐silencing methods. © 2015 by John Wiley & Sons, Inc.

Keywords: cell signaling pathways; mitochondria; oxidative stress; protein phosphorylation and p66Shc protein

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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Mitochondrial and Cytosolic Fractions
  • Basic Protocol 2: Isolation of Mitochondrial‐Associated Membranes
  • Basic Protocol 3: Immunoblotting of p66Shc and pSer36‐p66Shc in Different Fractions
  • Alternate Protocol 1: Immunoblotting for Activity of p66Shc Pathway‐Associated Proteins
  • Support Protocol 1: Pharmacological Inhibition of p66Shc Pathway
  • Support Protocol 2: Knockdown of p66Shc in Cell Lines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Isolation of Mitochondrial and Cytosolic Fractions

  • Cells of interest
  • Appropriate growth medium for cells, containing 10% fetal bovine serum (FBS)
  • Phosphate buffered saline (PBS; appendix 2A)
  • 0.05% (w/v) trypsin‐EDTA (Invitrogen, cat. no. 25300‐062)
  • Homogenization medium (see recipe)
  • Mitochondria isolation medium (see recipe)
  • Mitochondria resuspension buffer (see recipe)
  • 10‐cm2 cell culture dishes
  • 15‐ml conical polypropylene tubes (e.g., Corning Falcon)
  • Refrigerated low‐speed tabletop centrifuge (e.g., Sigma Model 2K15)
  • Stirrer motor with electronic speed controller
  • Motor‐driven tightly fitting glass/Teflon Potter‐ Elvehjem homogenizer
  • 30‐ml polypropylene centrifugation tubes
  • Ultracentrifuge (e.g., Beckman Coulter Optima L‐100 XP)
  • Additional reagents and equipment for pharmacological inhibition ( protocol 5) or knockdown ( protocol 6) of p66She pathway

Basic Protocol 2: Isolation of Mitochondrial‐Associated Membranes

  • Crude mitochondrial fraction ( protocol 1)
  • MAM isolation buffer I (see recipe)
  • Mitochondria resuspension buffer (see recipe)
  • Ultra‐Clear 14‐ml polyallomer ultracentrifuge tubes
  • Ultracentrifuge: e.g., Beckman Coulter Optima L‐100 XP with, e.g., SW 40 Ti rotor, swinging‐bucket, 6 × 14 ml, and Beckman Type 70 Ti rotor, fixed‐angle, 8 × 39 ml
  • Refrigerated low‐speed centrifuge
  • 30‐ml tubes for refrigerated low‐speed centrifuge
  • Polycarbonate tubes with cap assembly (Beckman, cat. no. 355618, for use with 70Ti rotor)

Basic Protocol 3: Immunoblotting of p66Shc and pSer36‐p66Shc in Different Fractions

  • Mitochondrial, cytosolic, and MAM fractions (see Basic Protocols protocol 11 and protocol 22, respectively)
  • 10× Cell Lysis Buffer (Cell Signaling, cat. no. 1679803S)
  • Phenylmethylsulfonyl fluoride (PMSF)
  • Protease and phosphatase inhibitors (Sigma‐Aldrich, cat. no. P8340 and P5726, respectively)
  • Bradford reagent (see recipe)
  • 6× Laemmli buffer (see recipe)
  • 10× SDS‐PAGE running buffer (see recipe)
  • Standard protein marker (e.g., Precision Plus Protein Dual Color Standard; BioRad, cat. no. 161‐0374)
  • Methanol
  • 10× transfer buffer (see recipe)
  • Bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A6003)
  • 10× Tris‐buffered saline/Tween 20 (TBST; see recipe)
  • Mouse monoclonal anti‐SHC/p66 – pSER36 (Calbiochem, cat. no. 566807)
  • Goat anti‐mouse IgG‐AP (Santa Cruz Biotechnology, cat. no. sc‐2008)
  • ECF substrate (Thermo Fisher Scientific, cat. no. RPN3685)
  • Mouse monoclonal anti‐SHC (BD Bioscience, cat. no. 610879)
  • Anti‐actin antibody, clone C4 (Millipore, cat. no. MAB1501)
  • 15‐ and 50‐ml disposable polystyrene tubes (e.g., BD Falcon)
  • 0.5‐ and 1.5 ml microcentrifuge tubes (e.g., VWR)
  • Accublock digital dry bath (LabNet international, Inc.)
  • Polyvinylidene difluoride (PVDF) membrane (Millipore, cat. no. IPVH00010) or nitrocellulose membrane (also see Gallagher et al., )
  • Mini Trans‐Blot Cell (BioRad, cat. no. 170‐3930)
  • Shaker plate
  • BioSpectrum multispectral imaging system (UVP)
  • Absorbance plate reader (e.g., Victor X3 Multilabel Reader, PerkinElmer)
  • Computer running Microsoft Excel, Quantity One 4.6.6 (BioRad), NIH ImageJ, and statistical software analysis
  • Additional reagents and equipment for protein assay ( appendix 3I; Krohn, ), SDS–polyacrylamide gel electrophoresis ( appendix 3F; Gallagher, ), immunoblotting (Gallagher et al., )

Alternate Protocol 1: Immunoblotting for Activity of p66Shc Pathway‐Associated Proteins

  Additional Materials (also see protocol 3)
  • Antibodies against p66Shc‐associated proteins of interest (Table 25.6.2)
Table 5.6.2   Additional Materials (also see protocol 3)Examples of Antibodies to Use in the Alternate Protocol

Protein/antibody against Vendor Catalog number
Bim Cell Signaling 2819
Forkhead box O3 (FoxO3a) Abcam ab4709
Forkhead box O3 (FoxO3a, phosphoSer253) Abcam ab47285
Pin1 Millipore 07‐091
Protein kinase B (PKB) or AKT (phosphoSer473) Cell Signaling 9271
Protein kinase B (PKB) or AKT Cell Signaling 9272
Protein kinase Cβ (PKCβ) Santa Cruz Biotechnology sc‐210
Protein phosphatase 2 (PP2A) Millipore 05‐421
Ras Cell Signaling 3965
Superoxide dismutase 1 (SOD 1) Santa Cruz Biotechnology sc‐11407
Superoxide dismutase 2 (SOD 2) Abcam ab13533

Support Protocol 1: Pharmacological Inhibition of p66Shc Pathway

  • Cells of interest (e.g., H9c2 cardiomyoblasts, ATCC‐CRL‐1446)
  • High‐glucose DMEM (Sigma‐Aldrich, cat. no. D‐5648) containing 10% FBS (Invitrogen, cat. no. 16000‐044), 1× penicillin‐streptomycin (Invitrogen, cat. no. 15140‐122) and 1.5 g/liter sodium bicarbonate (Sigma‐Aldrich, cat. no. S5761)
  • Xenobiotic(s) of interest
  • 10‐cm2 cell culture dishes
  • 10 mM hispidin stock solution (see recipe)
  • Dimethyl sulfoxide (DMSO; Sigma‐Aldrich, cat. no. W387509)

Support Protocol 2: Knockdown of p66Shc in Cell Lines

  • Cell line of interest
  • Appropriate medium for cell line of interest
  • p66Shc‐constructs (siRNA, shRNA)
  • Constructs with random sequence
  • Opti‐MEM medium (Invitrogen, cat. no. 11058‐021)
  • Lipofectamine 2000 (Invitrogen, cat. no. 11668‐019)
  • 24‐well cell culture plates
  • 0.5 ml Eppendorf microcentrifuge test tubes (e.g., VWR)
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