Fig. 3

NF-YAx substitutes NF-YAl in NF-Y but does not bind Sp1. a EMSA demonstrating specific CCAAT-box binding NF-Y complexes (arrow) formed by in vitro translated NF-YAl, NF-YAs or NF-YAx, combined with NF-YB and NF-YC (left panel) plus competition EMSA showing inhibition of specific NF-Y binding complexes (arrow) in the presence of excess cold specific (S-oligo) but not non-specific (NS-oligo) oligonucleotide, and anti-NF-YB and anti-NF-YA antibodies but not pre-immune IgGs (IgG) (right panel). b EMSA showing changes in NF-Y electrophoretic mobility (arrows) under conditions of constant NF-YAl and increasing NF-YAx levels and vice versa, plus NF-YB and C, demonstrating reciprocal NF-YAl and NF-YAx substitution in NF-Y complexes. c Western blot demonstrating NF-YAl, NF-YAs, NF-YAx and β-actin levels in stable NF-YAl, NF-YAs and NF-YAx1 SH-SY5Y-transfectant extracts (30 μg) (left panel) plus d IF demonstration of nuclear NF-YAl, NF-YAs and NF-YAx localization in stable NF-YAl, NF-YAs and NF-YAx1-SH-SY5Y transfectants (bar = 50 μm). e EMSA of specific CCAAT-box binding NF-Y complexes (arrow) in stable NF-YAl, NF-YAs, NF-YAx1 and NF-YA× 2 SH-SY5Y-transfectant nuclear extracts, with competition EMSA demonstrating specificity by inhibition with cold specific (S) but not non-specific (NS) oligonucleotides. f IP/Western blots showing NF-YA isoform levels in SL2 extracts (30 μg), 72 h following co-transfection with pAc SP1 and NF-YA insect expression vectors (1st panel); Sp1 and IgG levels in Sp1 immunoprecipitates (500 μg SL2 extracts) (2nd panel); Sp1 pull down of NF-YAl and NF-YAs (red asterisks) but not NF-YAx in Sp1 immunoprecipitates (500 μg SL2 extracts) (3rd panel), and the absence of Sp1, NF-YAl, NF-YAs and NF-YAx in pre-immune IgG precipitates (500 μg SL2 extracts) (4th and 5th panels). g IP/ Western blots of NF-YA isoform and β-actin protein levels in stable NF-YAl, NF-YAs and NF-YAx1 SH-SY5Y-transfectant extracts (30 μg) (1st panel); Sp1 and IgG levels in Sp1 immunoprecipiates (500 μg extracts) (2nd panel); Sp1 pulldown of NF-YAl and NF-YAs (*) (overlapping NS1 and NS2 bands) but not NF-YAx (arrow) (3rd panel), and the absence of Sp1, NF-YAl, NF-YAs and NF-YAx but not non-specific NS1, NS2 and NS3 bands in pre-immune immunoprecipitates (500 μg of each extract) (4th and 5th panels)