Gfp fl

For western blotting, cells were lyzed in EBC₂₅₀ lysis buffer. Whole-cell lysates were separated by SDS–PAGE, transferred to PVDF membranes, and hybridized to an appropriate primary antibody and HRP-conjugated secondary antibody for subsequent detection by ECL. Antibodies used include actin (C-11, Santa Cruz Biotechnology), Ac-p53-K382 (2525P; Calbiochem, La Joya, CA, USA), GFP (FL; Santa Cruz Biotechnology), p21^CIP1 (SXM-30; BD Pharmingen, San Diego, CA, USA), p53 (DO-1, Santa Cruz Biotechnology), phospho-Akt (Cell Signaling Technology, Danvers, MA, USA), SIRT1 (07-131, Upstate Biotechnology, Lake Pacid, NY, USA), and PAI-1 (D9C4, Cell Signaling Technology). For IP analyses, cells were lyzed in EBC₁₅₀ buffer. Whole-cell lysates were precleared with protein A bead slurry (Upstate Biotechnology), incubated with an appropriate antibody at 4 °C overnight, and subsequently captured with protein A bead slurry (Upstate Biotechnology) for 3 h. Immunoprecipitates were subjected to western blot analyses.

Rabbit polyclonal antibodies against c-myc (A-14) and GFP (FL) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against GAPDH were purchased from Proteintech. Mouse monoclonal antibodies against vinculin, clone hVIN-1, were purchased from Sigma Aldrich (St. Louis, MO, USA). Mouse monoclonal antibodies against enterovirus VP1 (NCL-entero) were purchased from Novocastra (Buffalo Grove, IL, USA). XtremeGene HP plasmid transfection reagent was purchased from Sigma Aldrich. Brefeldin A (Invitrogen, Carlsbad, CA, USA) was dissolved in DMSO and used at a final concentration of 5 µg/mL. Also, 2-aminoethoxydiphenylborane (2APB, Sigma Aldrich) was diluted in DMSO and used at a final concentration of 100 µM.