Figure 2

Characterization of X box motifs in promoter regions of pachytene-expressed genes. (A). Examples of gel shift results using oligos for four different X boxes and a whole cell extract from pachytene spermatocytes. In each case one lane shows the super-shift bands resulting from pre-incubation with a specific RFX2 antibody. The RFX2 heteromer band "ht" is likely formed by an RFX1:RFX2 dimer [11, 13]. The identity of the prominent bottom band "ns", which is not prevalent in nuclear extracts, is unknown. (B). Examples of gel shift competition in which the labeled probe was IL5RA [32], and cold competitor oligos were added to reactions in amounts from 5 to 50 ng. Spag6 is a strong binding site while Dkkl1 is relatively weak. (C). Quantitative display of the results of gel shift competition. The RFX2 homodimer band was quantitated by phosphorimaging with results normalized to the bound complex obtained with no competitor. Points are the average of two independent experiments. An oligo for the unrelated NFY binding site did not compete over the same concentration range (not shown).