Figure 3

Mislocalization of NF-YA in tuberin-deficient cells. A. TSC2^-/- and TSC2^+/+ cells were plated on a 2-well chamber slide. Cells were fixed then immunostained with primary anti-NF-YA antibody and secondary anti-goat IgG labeled with Alexa Fluor 488 (red fluorescence). The same cells were also incubated with DAPI (blue fluorescence, same microscopic fields) to identify nuclei. Overlay of NF-YA and DNA staining, demonstrating nuclear localization of NF-YA in the wild type and tuberin-deficient cells infected with adenovirus expressing tuberin. B. Nuclear and cytoplasmic fraction proteins were extracted from Tsc2 ^-/- cells non-infected and infected with adenovirus expressing tuberin. Immunoblot analysis of tuberin and NF-YA in the nuclear and cytoplasmic fractions was analyzed as described in Method section. Lamin B and actin antibodies were used as nuclear and cytoplasmic markers, respectively.