Nonphosphorylated Sp1 has higher DNA binding activity to the cyclin D3 promoter. A cyclin D3 DNA fragment that extends from −190 to −99 bp was used in a gel mobility shift assay. (A) Before being used in the gel mobility shift assay, human recombinant Sp1 (hrSp1) was subjected to dephosphorylation by protein phosphatase 1 (PP1) in the absence or presence of PP1 inhibitor 2 (I2) or to a phosphorylation reaction with casein kinase II (CKII), under conditions shown before to indeed affect the phosphorylation state of SP1 (as in Fig 8). (B) Nuclear extracts from Y10 cells were subjected to a kinase reaction with CKII, before being used in the gel mobility shift assay. (C) Nuclear extracts from Y10 cells were subjected to dephosphorylation with PP1 in the absence or presence of I2, before being used in the gel mobility shift assay. The buffer required for PP1 activity is different from that for CKII activity (see Materials and Methods), thus resulting in a somewhat different intensity of Sp1 binding under the two buffer conditions. The arrow points to the Sp1 complex, initially confirmed by supershift experiments with anti-Sp1 (as in Fig 5).