Cell Biol. EBP50 in Akt-dependent cell proliferation. (23). These observations suggest that EBP50 is usually a critical regulator of Skp2 and consequently cell proliferation. Yet, the mechanisms by which this regulation occurs are not known. Here we describe a novel phosphorylation-dependent conversation between Skp2 and EBP50 that controls Skp2 localization, stability, and function. EXPERIMENTAL PROCEDURES Plasmids and Mutagenesis The plasmid encoding N-terminal Flag-human EBP50 was described previously (25). The mutants S1, S2, ERM, S1/S2, and T156A EBP50 constructs were made from Flag-EBP50 by using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). [L424A]Skp2 mutant construct was also made by mutagenesis from Flag-tagged Skp2 (a gift from Dr. Michele Pagano, New York University School of Medicine). All plasmid DNA sequences were confirmed by sequence analysis (GeneWiz). Cell Culture and Transfection Primary VSMC were isolated from murine thoracic aortic explants and cultured in Dulbecco’s modified eagle media (DMEM) made up of 10% fetal bovine serum (FBS) in 5% CO2 at 37 C. All experiments were performed with cells between passages 3 and 15. Cells were transfected with siRNA for Skp2 (0.1 m) using DharmaFECT Duo transfection reagent (Dharmacon, Thermo Scientific) and used for experiments 72 h after transfection. YFP-tagged and Flag-tagged EBP50 constructs, Flag-tagged Skp2, and constitutively active Myr-Akt (a gift from Dr. Daniel Altschuler, University of Pittsburgh School of Medicine) were introduced in primary VSMC (1 106) by electroporation using an AMAXA electroporator and the Basic Nucleofect kit for primary easy muscle cells (Lonza). CHO cells were cultured in Ham’s Rabbit Polyclonal to STK36 F-12 medium supplemented with 10% FBS. EBP50 constructs and Myc-Akt were transfected in CHO cells using Fugene6 (Promega). Immunofluorescence Cells on glass coverslips were fixed with 4% paraformaldehyde and incubated with blocking buffer made up of 5% goat serum and 0.2% Nonidet P-40 (Nonidet P-40) in PBS. Primary rabbit anti-Skp2 (Santa Cruz Biotechnology, 1:1000) or anti-Flag (Santa Cruz Biotechnology, 1:500) were applied in the same buffer overnight at 4 C. Coverslips were washed with PBS, incubated with Alexa546-conjugated anti-rabbit secondary antibody (Molecular Probes, 1:1000) and 4,6-diamidino-2-phenylindole (DAPI, 0.1 g/ml; ML167 Sigma) for 2 h and washed again. Coverslips were mounted for immunofluorescence microscopy and analyzed with an Olympus Fluoview confocal laser-scanning microscope with an 63 oil immersion objective. Image analysis was performed with ImageJ software (National Institutes of Health). Western Blot Analysis Cells were lysed in urea lysis buffer (4 m urea, 62.5 mm Tris, 2% SDS, 1 mm EDTA) made up of a ML167 protease inhibitor mixture. The cell lysates were resolved by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes, which were then subjected to two sequential incubations with appropriate primary antibodies (1:500 dilution for EBP50, p21cip1, and 1:1000 dilution for Skp2 (all from Santa Cruz Biotechnology); 1:1000 dilution for p27kip1, pAkt, Akt antibodies (Cell Signaling); 1:5000 dilution for actin (Sigma)) and horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (1:2000, Cell Signaling). Immunoreactivity was detected by incubation with Immune-Star ECL (Bio-Rad). Quantitation of band intensity was performed with the Image J software (National Institutes of Health). For co-immunoprecipitation assay, cells were lysed in RIPA buffer (Santa Cruz Biotechnology) made up of protease inhibitor mixture. Lysates ML167 were incubated with the indicated antibodies overnight and ML167 with protein A/G beads for 2 h. Immuno-bead complexes were washed twice with Nonidet P-40 buffer (1 m Tris-base, ML167 150 mm NaCl, 5 mm EDTA, and 0.5% Nonidet P-40). Bound proteins were then released with 2 Laemmli.