Figure 1

EMSA analysis of KLF15 binding. (A) KLF15-ZF fusion protein. Lanes 1 and 2 show western blots of crude bacterial lysates at 1.5 and 2 hours following induction of fusion protein expression. Anti-GST antibodies detect a major band of the anticipated size and additional lower molecular weight bands. Lane 3, Coomassie stained gel of affinity purified KLF15-ZF-GST fusion protein showing enrichment of major band containing full length fusion protein. (B) EMSA using ³²P-labeled oligomers (bRho29) containing a 29 bp fragment from bovine rhodopsin promoter (-94 to -66). Lane 1: no protein; Lane 2: 67.5 ng GST; Lane 3–5: 100, 50, 25 ng KLF15-ZF-GST. (C) Same as (B) except ³²P-labeled oligonucleotide (hRho29) contained corresponding sequence from human rhodopsin promoter. (D) Supershift using ³²P-bRho29 oligonucleotide and anti-KLF15 antibodies. Lane 1, no protein; Lane 2, 33 ng GST protein; Lane 3, 33 ng GST + anti-KLF15; Lane 4, 50 ng KLF15-ZF-GST protein; Lanes 5–7, 50 ng KLF15-ZF-GST protein plus increasing amounts of anti-KLF15. (E) Supershift using ³²P-bRho29 oligonucleotide and anti-GST antibodies. Lane 1, 50 ng KLF15-ZF-GST protein; Lanes 2–4, 50 ng KLF15-ZF-GST protein plus increasing amounts of anti-GST. Large arrows, KLF15 shifted bands; small arrows, non-specific bands; arrowheads, supershifted bands.