Inhibition of Proteasome Activity Promotes the Correct Localization of Disease-Causing α-Sarcoglycan Mutants in HEK-293 Cells Constitutively Expressing β-, γ-, and δ-Sarcoglycan
2008; Elsevier BV; Volume: 173; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2008.071146
ISSN1525-2191
AutoresStefano Gastaldello, Simona D'Angelo, Susanna Franzoso, Francesco Mari, C. Angelini, Romeo Betto, Dorianna Sandonà,
Tópico(s)Cellular transport and secretion
ResumoSarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four proteins, α-, β-, γ-, and δ-sarcoglycan, which are elements of a key transmembrane complex of striated muscle. The proper assembly of the sarcoglycan complex represents a critical issue of sarcoglycanopathies, as several mutations severely perturb tetramer formation. Misfolded proteins are generally degraded through the cell's quality-control system; however, this can also lead to the removal of some functional polypeptides. To explore whether it is possible to rescue sarcoglycan mutants by preventing their degradation, we generated a heterologous cell system, based on human embryonic kidney (HEK) 293 cells, constitutively expressing three (β, γ, and δ) of the four sarcoglycans. In these βγδ-HEK cells, the lack of α-sarcoglycan prevented complex formation and cell surface localization, wheras the presence of α-sarcoglycan allowed maturation and targeting of the tetramer. As in muscles of sarcoglycanopathy patients, transfection of βγδ-HEK cells with disease-causing α-sarcoglycan mutants led to dramatic reduction of the mutated proteins and the absence of the complex from the cell surface. Proteasomal inhibition reduced the degradation of mutants and facilitated the assembly and targeting of the sarcoglycan complex to the plasma membrane. These data provide important insights for the potential development of pharmacological therapies for sarcoglycanopathies. Sarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four proteins, α-, β-, γ-, and δ-sarcoglycan, which are elements of a key transmembrane complex of striated muscle. The proper assembly of the sarcoglycan complex represents a critical issue of sarcoglycanopathies, as several mutations severely perturb tetramer formation. Misfolded proteins are generally degraded through the cell's quality-control system; however, this can also lead to the removal of some functional polypeptides. To explore whether it is possible to rescue sarcoglycan mutants by preventing their degradation, we generated a heterologous cell system, based on human embryonic kidney (HEK) 293 cells, constitutively expressing three (β, γ, and δ) of the four sarcoglycans. In these βγδ-HEK cells, the lack of α-sarcoglycan prevented complex formation and cell surface localization, wheras the presence of α-sarcoglycan allowed maturation and targeting of the tetramer. As in muscles of sarcoglycanopathy patients, transfection of βγδ-HEK cells with disease-causing α-sarcoglycan mutants led to dramatic reduction of the mutated proteins and the absence of the complex from the cell surface. Proteasomal inhibition reduced the degradation of mutants and facilitated the assembly and targeting of the sarcoglycan complex to the plasma membrane. These data provide important insights for the potential development of pharmacological therapies for sarcoglycanopathies. Mutations in sarcoglycans are responsible of autosomal recessive Limb-Girdle Muscular Dystrophy (LGMD) type 2C (γ-sarcoglycan), 2D (α-sarcoglycan), 2E (β-sarcoglycan), and 2F (δ-sarcoglycan), collectively named sarcoglycanopathies.1Roberds SL Leturcq F Allamand V Piccolo F Jeanpierre M Anderson RD Lim LE Lee JC Tomé FM Romero NB Fardeau M Beckmann JS Kaplan J-C Campbell KP Missense mutations in the adhalin gene linked to autosomal recessive muscular dystrophy.Cell. 1994; 78: 625-6330Abstract Full Text PDF PubMed Scopus (434) Google Scholar, 2Bonnemann CG Modi R Noguchi S Mizuno Y Yoshida M Gussoni E McNally EM Duggan DJ Angelini C Hoffmann EP Ozawa E Kunkel LM β-Sarcoglycan mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex.Nat Genet. 1995; 11: 266-273Crossref PubMed Scopus (426) Google Scholar, 3Noguchi S McNally EM Ben Othmane K Hagiwara Y Mizuno Y Yoshida M Yamamoto H Bonnemann CG Gussoni E Denton PH Kyriakides T Middleton L Hentati F Ben Hamida V Nonaka I Vance JM Kunkel LM Ozawa E Mutations in the dystrophin-associated protein γ-sarcoglycan in chromosome 13 muscular dystrophy.Science. 1995; 270: 819-822Crossref PubMed Scopus (479) Google Scholar, 4Nigro V Moreira EDS Piluso G Vainzof M Belsito A Politano L Puca AA Passos-Bueno MR Zatz M Identification of a novel sarcoglycan gene at 5q33 encoding a sarcolemmal 35 kDa glycoprotein.Nat Genet. 1996; 14: 195-198Crossref PubMed Scopus (388) Google Scholar These disorders are characterized by the progressive wasting of skeletal muscle with predominant involvement of the pelvic and shoulder girdle musculature.5Ozawa E Mizuno Y Hagiwara Y Sasaoka T Yoshida M Molecular and cell biology of the sarcoglycan complex.Muscle Nerve. 2005; 32: 563-576Crossref PubMed Scopus (130) Google Scholar In muscle membrane, the four sarcoglycans form a subcomplex closely associated to a major complex comprising dystrophin, the gene product of Duchenne and Becker Muscular Dystrophy, dystroglycans (α and β), dystrobrevins, syntrophins, and sarcospan.6Blake DJ Weir A Newey SE Davies K Function and genetics of dystrophin and dystrophin-related proteins in muscle.Physiol Rev. 2002; 82: 291-329Crossref PubMed Scopus (937) Google Scholar This multimeric complex, known as the dystrophin glycoproteins complex (DGC), provides a physical linkage between the actin cytoskeleton and the extracellular matrix7Pasternak C Wong S Elson EL Mechanical function of dystrophin in muscle cells.J Cell Biol. 1995; 128: 355-361Crossref PubMed Scopus (244) Google Scholar and is essential to protect muscle membrane integrity during contraction. 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Besides the role in providing membrane stability, recent evidence indicates that sarcoglycans could also be involved in signal transduction. In fact, it has been proposed that the sarcoglycan complex could participate in bidirectional signaling with integrins,18Yoshida T Pan Y Hanada H Iwata Y Shigekawa A Bidirectional signaling between sarcoglycans and the integrin adhesion system in cultured L6 myocytes.J Biol Chem. 1998; 273: 1583-1590Crossref PubMed Scopus (120) Google Scholar link filamin-2 in cytoskeletal signaling,19Thompson TG Chan YM Hack AA Brosius M Rajala M Lidov HG McNally EM Watkins S Kunkel LM Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein.J Cell Biol. 2000; 148: 115-126Crossref PubMed Scopus (244) Google Scholar and provide an anchorage for neuronal nitric oxide synthase.20Crosbie RH Barresi R Campbell KP Loss of sarcolemma nNOS in sarcoglycan-deficient muscle.FASEB J. 2002; 16: 1786-1791Crossref PubMed Scopus (73) Google Scholar Recently, it has been shown that the cytoplasmic tail of γ-sarcoglycan is phosphorylated after mechanical stimulation.21Barton ER Impact of sarcoglycan complex on mechanical signal transduction in murine skeletal muscle.Am J Physiol. 2006; 290: C411-C419Crossref Scopus (69) Google Scholar Lastly, α-sarcoglycan possesses an ecto-ATPase activity,22Betto R Senter L Ceoldo S Tarricone E Biral D Salviati G Ecto-ATPase activity of α-sarcoglycan (adhalin).J Biol Chem. 1999; 19: 7907-7912Crossref Scopus (74) Google Scholar, 23Sandonà D Gastaldello S Martinello T Betto R Characterization of the ATP-hydrolyzing activity of α-sarcoglycan.Biochem J. 2004; 381: 105-112Crossref PubMed Scopus (34) Google Scholar which could play a role in the extracellular ATP-dependent modulation of skeletal muscle contractility.24Sandonà D Danieli-Betto D Germinario E Biral D Martinello T Gastaldello S Betto R The T-tubule membrane ATP-operated P2X4 receptor influences contractility of skeletal muscle.FASEB J. 2005; 19: 1184-1186PubMed Google Scholar Studies on the assembly of the sarcoglycan complex, during the early stage of myotube differentiation, have provided evidence that sarcoglycans are co-translationally translocated in the endoplasmic reticulum (ER), where they associate during the transport through the Golgi to the plasma membrane.25Hack AA Lam MY Cordier L Shoturma DI Ly CT Hadhazy MA Hadhazy MR Sweeney HL McNally EM Differential requirement for individual sarcoglycans and dystrophin in the assembly and function of the dystrophin-glycoprotein complex.J Cell Sci. 2000; 113: 2535-2544PubMed Google Scholar, 26Noguchi S Wakabayashi E Imamura M Yoshida M Ozawa E Formation of sarcoglycan complex with differentiation in cultured myocytes.Eur J Biochem. 2000; 267: 640-648Crossref PubMed Scopus (56) Google Scholar Organization of the sarcoglycan complex occurs in a strict equimolar stoichiometry,27Jung D Duclos F Apostol B Straub V Lee JC Allamand V Venzke DP Sunada Y Moomaw CR Leveille CJ Slaughter CA Crawford TO McPherson JD Campbell KP Characterization of δ-sarcoglycan, a novel component of the oligomeric sarcoglycan complex involved in limb-girdle muscular dystrophy.J Biol Chem. 1996; 271: 32321-32329Crossref PubMed Scopus (84) Google Scholar a ratio that appears to be mandatory, because overexpression of γ-sarcoglycan in mice causes muscular dystrophy.28Zhu X Hadhazy M Groh ME Wheeler MT Wollmann R McNally EM Over-expression of γ-sarcoglycan induces severe muscular dystrophy. 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The frequency of sarcoglycanopathy among cases of autosomal recessive LGMD varies worldwide, with some regional differences. For instance, sarcoglycanopathy is the prevailing autosomal recessive LGMD form in the Brazilian population (68%),32Vainzof M Passos-Bueno MR Pavanello RC Marie SK Oliveira AS Zatz M Sarcoglycanopathies are responsible for 68% of severe autosomal recessive limb-girdle muscular dystrophy in the Brazilian population.J Neurol Sci. 1999; 164: 44-49Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar and in India (54%),33Meena AK Sreenivas D Sundaram C Rajasekhar R Sita JS Borgohain R Suvarna A Kaul S Sarcoglycanopathies: a clinico-pathological study.Neurol India. 2007; 55: 117-121Crossref PubMed Scopus (22) Google Scholar whereas it represents 49% in the United States.12Moore S Shilling CJ Westra S Wall CRN Wicklund MP Stolle C Brown CA Michele DE Piccolo F Winder TL Stence A Barresi R King N King W Florence J Campbell KP Fenichel GM Stedman HH Kissel JT Griggs RC Pandya S Mathews KD Pestronk A Serrano C Darvish D Mendell JR Limb-girdle muscular dystrophy in the United States.J Neuropath Exp Neurol. 2006; 65: 995-1003Crossref PubMed Scopus (140) Google Scholar In other countries, such as Australia and Italy, the frequency of sarcoglycanopathy is lower (below 20%).9Boito C Fanin M Siciliano G Angelini C Pegoraro E Novel sarcoglycan gene mutations in a large cohort of Italian patients.J Med Genet. 2003; 40: e67Crossref PubMed Scopus (31) Google Scholar, 34Lo HP Cooper ST Evesson FJ Seto JT Chiotis M Tay V Compton AG Cairns AG Corbett A MacArthur DG Yang N Reardon K North KN Limb–girdle muscular dystrophy: diagnostic evaluation, frequency and clues to pathogenesis.Neuromuscul Disord. 2008; 18: 34-44Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 35Guglieri M Magri F D'Angelo MG Prelle A Morandi L Rodolico C Cagliani R Mora M Fortunato F Bordoni A Del Bo R Ghezzi S Pagliarani S Lucchiari S Salani S Zecca C Lamperti C Ronchi D Aguennouz M Ciscato P Di Blasi C Ruggieri A Moroni I Turconi A Toscano A Moggio M Bresolin N Comi GP Clinical, molecular, and protein correlations in a large sample of genetically diagnosed Italian limb girdle muscular dystrophy patients.Hum Mutat. 2008; 29: 258-266Crossref PubMed Scopus (148) Google Scholar In Europe, North America, Brazil, and India the majority of patients deficient for sarcoglycan proteins has genetic defects in α-sarcoglycan (LGMD-2D), a form less frequent in Northern Africa.9Boito C Fanin M Siciliano G Angelini C Pegoraro E Novel sarcoglycan gene mutations in a large cohort of Italian patients.J Med Genet. 2003; 40: e67Crossref PubMed Scopus (31) Google Scholar, 12Moore S Shilling CJ Westra S Wall CRN Wicklund MP Stolle C Brown CA Michele DE Piccolo F Winder TL Stence A Barresi R King N King W Florence J Campbell KP Fenichel GM Stedman HH Kissel JT Griggs RC Pandya S Mathews KD Pestronk A Serrano C Darvish D Mendell JR Limb-girdle muscular dystrophy in the United States.J Neuropath Exp Neurol. 2006; 65: 995-1003Crossref PubMed Scopus (140) Google Scholar, 33Meena AK Sreenivas D Sundaram C Rajasekhar R Sita JS Borgohain R Suvarna A Kaul S Sarcoglycanopathies: a clinico-pathological study.Neurol India. 2007; 55: 117-121Crossref PubMed Scopus (22) Google Scholar, 36Carrie A Piccolo F Leturcq F de Toma C Azibi K Beldjord C Vallat JM Merlini L Voit T Sewry C Urtizberea JA Romero N Tomè FM Fardeau M Sunada Y Campbell KP Kaplan JC Jeanpierre M Mutational diversity and hot spots in the α-sarcoglycan gene in autosomal recessive muscular dystrophy (LGMD2D).J Med Genet. 1997; 34: 470-475Crossref PubMed Google Scholar, 37Duggan DJ Gorospe JR Fanin M Hoffman EP Angelini C Mutations in the sarcoglycan genes in patients with myopathy.N Engl J Med. 1997; 336: 618-624Crossref PubMed Scopus (199) Google Scholar, 38Trabelsi M Kavian N Daoud F Commere V Deburgrave N Beugnet C Llense S Barbot JC Vasson A Kaplan JC Leturcq F Chelly J Revised spectrum of mutations in sarcoglycanopathies.Eur J Hum Genet. 2008; ([Epub ahead of print])PubMed Google Scholar Analyses of muscle biopsies from LGMD-2D patients carrying α-sarcoglycan mutations reveal the absence or severe reduction of all four sarcoglycan subunits. According to the SGCA gene variant database (Leiden Muscular Dystrophy pages at http://www.DMD.nl), 47 sequence variants in the coding region of α-sarcoglycan have been reported to cause LGMD-2D, with the R77C missense substitution being the most frequently reported mutation.39Fokkema IF den Dunnen JT Taschner PE LOVD: easy creation of a locus-specific sequence variation database using an “LSDB-in-a-box” approach.Human Mutat. 2005; 26: 63-68Crossref PubMed Scopus (223) Google Scholar Thirty-five α-sarcoglycan missense mutations generate a complete protein with a single residue substitution, four nonsense mutations produce a truncated protein, the remaining eight (nucleotides duplication, deletion, or insertions) result in an incomplete/anomalous protein.39Fokkema IF den Dunnen JT Taschner PE LOVD: easy creation of a locus-specific sequence variation database using an “LSDB-in-a-box” approach.Human Mutat. 2005; 26: 63-68Crossref PubMed Scopus (223) Google Scholar It is possible that missense mutations in α-sarcoglycan produce a polypeptide that hampers the assembly of a stable sarcoglycan complex, leading to loss of all four sarcoglycans. Notably, all α-sarcoglycan missense mutations are mapped in the extracellular domain, a region critical for the organization of a stable sarcoglycan tetramer.31Chen J Shi W Zhang Y Sokol R Cai H Lun M Moore BF Farber MJ Stepanchick JS Bonnemann CG Chan YM Identification of functional domains in sarcoglycans essential for their interaction and plasma membrane targeting.Exp Cell Res. 2006; 312: 1610-1625Crossref PubMed Scopus (31) Google Scholar, 40Holt KH Campbell KP Assembly of the sarcoglycan complex. Insights for muscular dystrophy.J Biol Chem. 1998; 273: 34667-34670Crossref PubMed Scopus (107) Google Scholar Most of the missense mutations generate polypeptides that usually are unable to fold correctly. Misfolded, or damaged proteins typically undergo degradation via the ubiquitin-proteasome system.41Goldberg AL Protein degradation and protection against misfolded or damaged proteins.Nature. 2003; 426: 895-899Crossref PubMed Scopus (1713) Google Scholar However, some of the mutant proteins eliminated by the “cell's quality-control system” might still be functional if targeted to the correct cellular location. An example is the most common mutation of the cystic fibrosis transmembrane conductance regulator gene, ΔF508, resulting in a misfolded protein retained in the ER to be eventually targeted to degradation through proteasome.42Ward CL Omura S Kopito RR Degradation of CFTR by the ubiquitin-proteasome pathway.Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1147) Google Scholar However, the misfolded ΔF508 protein was demonstrated to be functional when it is forced to reach the cell membrane.43Cheng SH Gregory RJ Marshall J Paul S Souza DW White GA O'Riordan CR Smith AE Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1502) Google Scholar We hypothesize that some of the α-sarcoglycan mutants in LGMD-2D might also follow a similar course. 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The treatment with proteasome inhibitors was not only able to prevent degradation of the short dystrophin polypeptide, but also permitted its targeting to the cell membrane.48Bonuccelli G Sotgia F Schubert W Park DS Frank PG Woodman SE Insabato L Cammer M Minetti C Lisanti MP Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins.Am J Pathol. 2003; 163: 1663-1675Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 49Bonucelli G Sotgia F Capozza F Gazzero E Minetti C Lisanti MP Localized treatment with a novel FDA-approved proteasome inhibitor blocks the degradation of dystrophin and dystrophin-associated proteins in mdx mice.Cell Cycle. 2007; 6: 1242-1248Crossref PubMed Scopus (70) Google Scholar Notably, all of the dystrophin-associated proteins, normally degraded in mdx muscle since unstable in the absence of dystrophin, were also detected in the cell membrane of the dystrophic muscle.48Bonuccelli G Sotgia F Schubert W Park DS Frank PG Woodman SE Insabato L Cammer M Minetti C Lisanti MP Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins.Am J Pathol. 2003; 163: 1663-1675Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 49Bonucelli G Sotgia F Capozza F Gazzero E Minetti C Lisanti MP Localized treatment with a novel FDA-approved proteasome inhibitor blocks the degradation of dystrophin and dystrophin-associated proteins in mdx mice.Cell Cycle. 2007; 6: 1242-1248Crossref PubMed Scopus (70) Google Scholar Importantly, treatment with the proteasomal inhibitor MG132 rescued defective dystrophin and the other DGC proteins in Duchenne and Becker Muscular Dystrophy explants.50Assereto S Stringara S Sotgia F Bonuccelli G Broccolini A Pedemonte M Traverso M Biancheri R Zara F Bruno C Lisanti MP Minetti C Pharmaceutical rescue of the dystrophin complex in Duchenne and Becker skeletal muscle explants by proteasomal inhibitor treatment.Am J Physiol. 2006; 290: C577-C582Crossref Scopus (49) Google Scholar The present study was aimed at investigating 1) the fate of disease-causing α-sarcoglycan mutants, 2) the involvement of the ubiquitin-proteasome system in their degradation, and 3) whether the proteasome could become a potential target for drug treatments able to rescue sarcoglycan complex at the cell membrane of dystrophic muscle. To answer these questions, we developed a cellular expression system that permitted us to demonstrate that protein mutants do not assemble into a regular tetramer but are degraded by proteasome. Importantly, by inhibiting proteasome activity, we show that it is possible to avoid the degradation of α-sarcoglycan mutants and promote their assembly and their targeting to the cell membrane. The cell model also appears to be a suitable high-throughput screening system for identification of molecules that are able to rescue α-sarcoglycan mutants. Last but not least, experiments performed in muscle explants from one LGMD-2D patient show that inhibition of proteasome permits the rescue of α-sarcoglycan mutants even in skeletal muscle fibers. Though preliminary, the results offer the encouraging premise of therapies for the treatment of sarcoglycanopathies in humans. Full-length human α-sarcoglycan cDNA was donated by M.L. Kunkel (Harvard University) while δ- and γ-sarcoglycan mouse cDNA by Yi-umo M. Chan (McColl-Lockwood Laboratory for Muscular Dystrophy Research, Department of Neurology, Carolinas Medical Center). Full-length mouse β-sarcoglycan was generously provided by RIKEN Genomic Research Group. The sarcoglycan cDNAs were cloned in pcDNA3 expression vector; a 6-His tag was added at the extracellular C-ter of β-sarcoglycan and at the intracellular C-ter of α-sarcoglycan;23Sandonà D Gastaldello S Martinello T Betto R Characterization of the ATP-hydrolyzing activity of α-sarcoglycan.Biochem J. 2004; 381: 105-112Crossref PubMed Scopus (34) Google Scholar cMyc was added to the extracellular C-ter of δ-sarcoglycan; and HA1 was fused at the extracellular C-ter of γ-sarcoglycan. To facilitate multiple construct transfection experiments, the pcDNA3 vector was shortened by removing the neomycin resistance gene. Point mutations in α-sarcoglycan were engineered by QuikChange site-directed mutagenesis kit according to the manufacturer's protocol (Stratagene, La Jolla, CA). All constructs were verified by sequencing. Human embryonic kidney 293 cells (HEK-293) were seeded in plastic tissue flasks at a density of about 25,000 cells/cm2 and grown to about 70% confluence in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) containing 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. HEK-293 cells were transfected by the calcium phosphate method as described.23Sandonà D Gastaldello S Martinello T Betto R Characterization of the ATP-hydrolyzing activity of α-sarcoglycan.Biochem J. 2004; 381: 105-112Crossref PubMed Scopus (34) Google Scholar To select HEK-293 clones stably co-expressing β-, γ-, and δ-sarcoglycan, equimolar concentration of β-, γ-, and δ-constructs, together with pBABE vector, were used. This vector, conferring puromycin-resistance, was used at 1:10 molar ratio with respect to the other plasmids. Puromycin-resistant clones were selected by growing cells in medium supplemented with 0.25 μg/ml puromycin. Western blot and densitometric analyses showed that only three out 51 clones expressed β-, γ-, and δ-sarcoglycan at about 1:1:1 stoichiometry. Clone # 10.5, named βγδ-HEK cells, was used in the described experiments. Cell viability was determined with the Promega CellTiter Blue assay (Promega, Madison, WI). Thirty-six hours after transfection, cells were incubated for 8 hours either with the cell-permeable proteasome inhibitor MG132 (Sigma), lactacystin (Sigma), both diluted in DMSO, or bortezomib (Velcade, Millennium Pharmaceutical) diluted in physiological solution. Then, cells were washed with phosphate-buffered saline (PBS) and lysed with the M-PER extraction buffer (Pierce, Rockford, IL) supplemented with the Complete protease inhibitor cocktail (Roche, Basel, Switzerland). Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE). In some experiments, after drug incubation, cell surface proteins were purified by biotinylation. Forty-eight hours after transfection, cells were washed three times with ice-cold PBS. About 3.5 × 106 cells were incubated with 0.25 mg/ml Sulfo-NHS-LC-Biotin (Pierce) in PBS for 30 minutes at 4°C. To remove the unreacted biotin, cells were washed three times for 5 minutes with the neutralizing solution (1M Tris-Cl, pH 7.5, 0.9% NaCl). The biotin-labeled cells were harvested and lysed in 500 μl of the M-PER extraction buffer supplemented with protease inhibitor cocktail. Lysate was c
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