蛋白质磷酸化与信号传导(2)

3. Wash cells once with 5 ml FACS staining buffer (1x PBS, 1% FBS, 0.09% NaN3). 4. Resuspend cells in staining buffer at 10 x 106 cells/ml.

5. Aliquot 100 μl of cells to each tube. Perform surface staining by adding fluorochrome-

conjugated monoclonal antibodies specific for cell surface antigens . Incubate in the dark for 30 minutes at room temperature.

6. Wash cells once with 3ml staining buffer, pellet by centrifugation (250 x g), and discard

supernatant.

7. Permeabilize cells by thoroughly resuspending in 0.5 ml cold 90% methanol. Incubate for

30 minutes on ice.

8. Wash once with 3 ml staining buffer, pellet by centrifugation (250 x g) for 5 minutes and

discard supernatant.

9. Perform intracellular staining by resuspending fixed/permeabilized cells in 100 μl of

staining buffer containing 2 μl phospho-specific Anti-Erk 1/2 antibodies（BD Pharmingen cat# 612358）. Incubate at room temperature for 1 hour in the dark.

10. Wash once with 5 ml 1x staining buffer, pellet by centrifugation (250 x g) for 5 minutes and

discard supernatant.

11. Cells were then incubated with 2 μl PE-conjugated Rat anti-mouse-IgG1 antibody（BD

Pharmingen cat# 550083）for 30 minutes at room temperature in the dark. 12. Wash once with 5 ml staining buffer. Resuspend in 0.5 ml staining buffer prior to flow

cytometric analysis.

13. Run on flow cytometer. Flow cytometry was done using a FACScan analyzer (Becton

Dickinson, San Jose, CA). An air-cooled argon laser operating at 15mW was used for excitation. Fluorescence signals were collected using bandpass filters centered at 530nm and 585nm, with fluorescence compensation set to suppress FITC spillover into the PE channel.

六、預期結果、注意事項：

Effects of PMA is similar in CD3 positive and negative cells. Expect to see 20-30 fold increase in pERK1/2 and 10-20 fold increase in pMEK with PMA activation. Use isotype controls or omitting primary antibodies if needed. The specific MEK inhibitor U0126 (Cell Signal Technology #9903), added at a final concentration of 10mM for 15 min at 37° prior to PMA treatment, blocks the activation of pERK1/2 but not pMEK.

蛋白质磷酸化与信号传导

七、Western Blots

Protein extracts were prepared from isolated lymphocytes using lysis buffer (1% Triton X-100, 0.1% SDS, 50mM Tris (pH 8.0) 150 mM NaCl, 1mM phenylmethylsulfonyl fluoride, 0.1mM NaVO4, 0.1mM benzamidine, 5mg/ml leupeptin, and 5mg/ml aprotinin). Samples containing 25mg of total protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes using the Mini Trans-Blot Electrophoresis Transfer Cell (Bio-Rad, Mississauga, ON). ERK1/2 activity was detected using the phospho-specific antibody obtained from BD Pharmingen. Secondary antibody containing the horseradish peroxidase detection system for chemiluminescence was used as recommended by the manufacturer.

References： 1.

Burkett PJ., Nelson BJ., et al. 2003 Intracellular analysis of activated cell signaling molecules through the use of phospho-specific antibodies and flow cytometry. Unpublished data, Pharmingen R&D. 2.

Ulrike R. Schwarz, Jörg Geiger, Ulrich Walter, Martin Eigenthaler. 2003 Flow Cytometry Analysis of Intracellular Protein Phosphorylation for the Assessment of Activating and Inhibitory Signal Transduction Pathways in Human Platelets Thrombosis and Haemostasis. A preprint can be obtained, please contact M. Eigenthaler by email. 3.

van Huijsduijnen RH, Bombrun A, Swinnen D. 2002 Selecting protein tyrosine phosphatases as drug targets. Drug Discov Today 7(19):1013-9 4.

Wilhelm S, Chien DS. 2002 BAY 43-9006: preclinical data. Curr Pharm Des 8(25):2255-7. Bayer Research Center, Bayer Corporation 5.

Yoganathan N, Yee A, Zhang Z, Leung D, Yan J, Fazli L, Kojic DL, Costello PC, Jabali M, Dedhar S, Sanghera. 2002 Integrin-linked kinase, a promising cancer therapeutic target: biochemical and biological properties. J. Pharmacol Ther 93(2-3):233-42. 6.

Sue Chow, Harshna Patel, and David W. Hedley. 2001 Measurement of MAP Kinase Activation by Flow Cytometry Using Phospho-Specific Antibodies to MEK and ERK: Potential for Pharmacodynamic Monitoring of Signal Transduction Inhibitors. Cytometry (Communications in Clinical Cytometry) 46:72–78 7.

Demetri GD. 2001 Targeting c-kit mutations in solid tumors: scientific rationale and novel therapeutic options. Semin Oncol 28(5 Suppl 17):19-26 8.

Emery JG, Ohlstein EH, Jaye M. 2001 Therapeutic modulation of transcription factor activity. Trends Pharmacol Sci 22(5):233-40 9.

Wilkinson MG, Millar JB. 2000 Control of the eukaryotic cell cycle by MAP kinase signaling pathways. FASEB J. Nov;14(14):2147-57

10. Yoganathan TN, Costello P, Chen X, Jabali M, Yan J, Leung D, Zhang Z, Yee A, Dedhar

蛋白质磷酸化与信号传导

S, Sanghera 2000 Integrin-linked kinase (ILK): a "hot" therapeutic target. J. Biochem Pharmacol Oct 15;60(8):1115-9

11. Lee JC, Kumar S, Griswold DE, Underwood DC, Votta BJ, Adams JL. 2000 Inhibition of

p38 MAP kinase as a therapeutic strategy. Immunopharmacology May;47(2-3):185-201 12. Ciardiello F, Caputo R, Bianco R, Damiano V, Pomatico G, De Placido S, Bianco AR,

Tortora G. 2000 Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor- selective tyrosine kinase inhibitor. Clin Cancer Res 6:2053-2063.

13. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE,

Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos JM. 1998 Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273:18623-18632.

蛋白质磷酸化与信号传导

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