The C/EBPα 30-kDa form is defective in DNA binding. (A, Top) EMSA was performed using a double-stranded C/EBP binding site from the human G-CSF receptor.¹⁶  Equal amounts of nuclear extracts from nontransfected K562 cells (lanes 2-3), 2 independent clones (nos. 29 and 32) expressing the 42-kDa C/EBPα protein (lanes 4-7), and 2 independent clones (nos. 3 and 7) expressing the 30-kDa C/EBPα protein (lanes 8-11) were used as a source of DNA binding proteins. Lane 1 contained probe only. In lanes 3, 5, 7, 9, and 11, 1 uL of a supershifting C/EBPα antibody was added. ss indicates supershifted complex; C/EBP, C/EBP complex; and X, nonspecific complex observed with this probe.^6,16  Ab indicates antibody. (A, Bottom) The same extracts as used in lanes 2, 4, 6, 8, and 10 in the top panel were used in an EMSA assay with an NFY probe⁵²  as a control for integrity and quantity of nuclear binding proteins. (B, Top) EMSA was performed as shown in panel A. Nuclear extracts from estrogen-receptor vector only (ER) transfected K562 cells (lanes 2-3) and clones expressing the 42-kDa C/EBPα protein (lanes 4-5), Δ1-70 (lanes 6-7), and the 30-kDa C/EBPα protein (lanes 9-10) were used as a source of DNA binding proteins. The amount of extract was adjusted according to the Western blot in the bottom panel so that the amount of C/EBPα-ER fusion protein was the same in each binding reaction. Lane 1 contained probe only and lane 8 is a blank lane. In lanes 3, 5, 7, and 10, 1 uL of a supershifting C/EBPα antibody was added. (B, Bottom) Western blot of transfected K562 extracts used in the top of panel B using a rabbit antiestrogen receptor antibody. * Indicates nonspecific bands. The specific C/EBPα-ER fusion protein bands were quantitated with a Phosphorimager (Amersham).