Materials and Reagents

Methionine-free medium DMEM (Sigma-Aldrich, catalog number: D0422 )
Fetal calf serum (Hyclone, catalog number: SV30160.03 )
Fetal bovine serum (FBS)
Penicillin/streptomycin/glutamine
Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 10010-056 )
Protein assay kit (DC Protein Assay Kit I-500) (Bio-Rad, catalog number: 0111EDU )
Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, catalog number: sc-2003 )
EasyTaq -[³⁵S]-Methionine, 5 mCi (185 MBq), stabilized aqueous solution (Perkinelmer, catalog number: NEG709A005MC )
Hybond ECL Nitrocellulose Membrane (Amersham, catalog number: RPN68D )
Kodak Biomax XAR film (Sigma-Aldrich, catalog number: F5763 )
Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, catalog number: WBKLS0500 )
Anti-MDM2 (Santa Cruz)
HEPES
NaCl
Glycerol
Triton X-100
MgCl₂
EGTA
Na₄P₂O₇
NaF
Aprotinin
Leupeptin
PMSF
Na₃VO₄
2-mercaptoethanol
Acrylamide
Bromophenol blue
Ammonium persulfate (APS)
Lysis buffer (see Recipes)
HNTG buffer (see Recipes)
Protein A or G agarose beads (see Recipes)
2x laemmli buffer (see Recipes)
SDS-polyacrylamide gel (see Recipes)
10x electrophoresis buffer (see Recipes)
1x transfer buffer (see Recipes)

Equipment

Centrifuges
Vortexer
Tissue culture hood and incubator
Radioactive material and room
Western-Blot apparatus
Developer
Hamilton syringe
Spectrophotometer
Rocker
T25 flask

Procedure

Metabolic labeling
1. Wash 1 x 10⁷ cells/sample in 30 ml methionine-free medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin/glutamine for 3 times.
2. Harvest cells for 60 min in 10 ml methionine-free medium in T25 flask.
3. Resuspend cells in 10 ml medium containing 250 uCi/sample [³⁵S]-methionine for 30 min.
4. Wash cells twice in 10 ml PBS and centrifuge them at 1,200 rpm for 10 min.
5. Lyse the cells with 500 μl of lysis buffer for 30 min by vortexing 15 sec every 5 min at 4 °C.
6. Centrifuge at 10,000 x g for 30 sec to pellet the DNA at 4 °C.
7. Determine the protein levels in the supernatant by DC Protein assay kit I.
8. Take same amount of protein extracts (about 1 mg) for immunoprecipitation after protein quantification.
Immunoprecipitation
1. Incubate equal amount of lysates onvernight at 4 °C by rotation with 3 μg antibody against protein of interest (here 30 μl of anti-MDM2 antibody) in eppendorf tubes.
2. Spin down the lysates (in order to collect all the drops in the cap after rotation).
3. Add 30 μl of Protein A/G PLUS-Agarose slurry volume by pipetting with tips (edge already cut off) (see Recipes 3) and incubate 2 h on a rocker at 4 °C.
4. Wash immunoprecipitates (beads) with 500 μl HNTG buffer for 4 times by centrifuging beads at 10, 000 x g for 30 sec at 4 °C.
5. At the end aspirate HNTG buffer with Hamilton syringe.
6. Add 30 μl of Laemmli buffer 2x on beads.
7. Boil the sample for 5 min.
Resolving protein of interest on SDS-polyacrylamide gel electrophoresis and autoradiography
1. Resolve the protein on SDS-polyacrylamide gel electrophoresis under denaturing conditions.
2. Transfer it onto nitrocellulose membrane and newly synthesized [³⁵S]methionine-protein will be visualized after exposure to X-AR films.
3. Verify the immunoprecipitation loading by incubating overnight with appropriate primary and secondary antibodies (see Western-blot protocol).
4. Detect the protein with a chemoluminecent HRP substrate detection kit.

Recipes

Lysis buffer
50 mM HEPES (pH 7.0)
150 mM NaCl
10% glycerol
1% Triton X-100
1.5 mM MgCl₂
1 mM EGTA
10 mM Na₄P₂O₇
+add extemporary
10 mM NaF
1 mM DTT
10 μg L^-1 aprotinin
10 μg L^-1 leupeptin
1 mM PMSF
1 mM Na₃VO₄
HNTG buffer
50 mM HEPES (pH 7.0)
10% glycerol
0.3% Triton X-100
150mM NaCl
1 mM NaVO₄
Protein A or G agarose beads
Wash the beads twice with PBS
Restore to 50% slurry with PBS
(It is recommended to cut the edge of the tip to pipet)
2x laemmli buffer
4% SDS
20% glycerol
10% 2-mercaptoethanol
0.004% bromophenol blue
0.125 M Tris-HCl
SDS-polyacrylamide gel
10% PAGE
H₂O 4 ml
30% Acrylamide 3.3 ml
1.5 M Tris (pH 8.8) 2.5 ml
10% SDS 0.1 ml
10% Ammonium persulfate (APS) 0.1 ml
10% TEMED 0.012 ml
STACKING
H₂O 5.6 ml
Acrylamide (30%) 1.7 ml
0.5M Tris (pH 8.8) 2.5 ml
10% SDS 0.1 ml
10% Ammonium persulfate (APS) 0.125 ml
10% TEMED 0. 015 ml
10x electrophoresis buffer
Glycine 144 g
Tris base 30 g
20% SDS 50 ml
H₂O qsp 1 L
1x transfer buffer
Glycine 14 g
Tris base 3 g
20% Ethanol 200 ml
H₂O qsp 1 L

Acknowledgments

The protocol was previously published in Nakatake et al. (2012). This work was supported by grants from ” Association pour la Recherche sur le Cancer (projet libre 2012), Agence Nationale de la Recherche, programme Jeunes Chercheuses et Jeunes Chercheurs, Laboratory of Excellence Globule Rouge-Excellence is funded by the program “Investissements d’avenir.” HS was supported by fellowships from la Ligue Nationale Contre le Cancer.

References

Nakatake, M., Monte-Mor, B., Debili, N., Casadevall, N., Ribrag, V., Solary, E., Vainchenker, W. and Plo, I. (2012). JAK2(V617F) negatively regulates p53 stabilization by enhancing MDM2 via La expression in myeloproliferative neoplasms. Oncogene 31(10): 1323-1333.