Accunanobead streptavidin magnetic nanobeads

pCMV–Myc–ATP synthase 5 alpha, pCMV–Myc–ATP synthase 5 beta, or pCMV–Myc–14–3–3ζ plasmids were transfected into HEK293T cells (CRL–11268; ATCC). Transfected cells were selected with 3 μM puromycin (BML-GR312-0050; Enzo Life Sciences, Farmingdale, NY, USA) for 14 days. During selection, the medium was changed every 4 day. After selection, cells were treated with 8 μM biotinylated KB1541 for 5 days. Cells were lysed with lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM KCl, 1 mM PMSF, 2 mM benzamidine, 0.05% NP-40, protease inhibitor cocktail (04693116001; Roche)]. Then, precipitation was performed using AccuNanoBead™ Streptavidin Magnetic Nanobeads (TA-1015-1; Bioneer). Proteins were separated on a 10% SDS–PAGE gel and analyzed by immunoblotting.

Senescent fibroblasts were treated for 14 days with 8 μM biotinylated KB1541. Immunoprecipitation was performed using AccuNanoBead™ Streptavidin Magnetic Nanobeads (TA-1015-1; Bioneer, Daejon, Korea). The eluted proteins were applied on to an Ion Mobility Tandem Mass Spectrometer/Time-of-flight mass spectrometry (IM–MS/MS TOF) (LCQ Deca XP-Plus, Thermo Finnigan, San Jose, CA, USA). Data analysis was performed as previously described [54 (link)].