Anti foxo1 c29h4

Splenocytes were pooled from 6 mice, and 5 × 10⁶ primary NK cells were purified from the splenocytes by using a mouse NK negative selection kit (Miltenyi Biotec), followed by FACS-sorting. Cell lysates were prepared using a NP40 cell lysis buffer, supplemented with 10 mM phenylmethanesulfonyl fluoride and 1× proteinase inhibitor cocktail (Sigma). An equal amount of protein was used in each immunoprecipitation, and was pre-cleared with 1 μg rabbit (for Foxo1 IP) or mouse (for Sp1 IP) IgG control antibodies plus 50 μl Dynabeads Protein A or Dynabeads Protein G, respectively, for 2 hours at 4 °C. The precleaned lysates were then incubated with anti-Foxo1 (C29H4) (1:100, Cell Signal Technology) or anti-Sp1 (E3) (5 μg, Santa Cruz Technology) antibodies at 4 °C overnight. An equal volume of cell lysate, incubated with the same species normal IgG, was taken as a control (Cell Signaling Technology or Abcam). The immunoprecipitates were subsequently incubated with 50 μl Dynabeads Protein A or Dynabeads Protein G for additional 4 hours at 4 °C, washed with lysis buffer, and subjected to immunoblotting against Foxo1 (C29H4) (Cell Signaling Technology) or Sp1 (Millipore). A Clean-Blot IP Detection Kit (HRP) (Pierce) was used as a secondary antibody.