FIGURE 8.

Binding of Sp family of transcription factors to the PKCδ promoter in NIE115 cells. A, ChIP assays in NIE115 cells indicate a physical association of Sp1, Sp3, and Sp4 with the PKCδ promoter region. Cross-linked chromatin was isolated from NIE115 cells transfected with the expression plasmids for Sp1 (pN3-Sp1), Sp3 (pN3-Sp3 FL), Sp4 (pN3-Sp4), or the empty vector pN3, as indicated. Isolated chromatin was enzymatically digested and immunoprecipitated with anti-Sp1 (lanes 2 and 7), anti-Sp3 (lanes 3 and 10), anti-Sp4 (lanes 4 and 13), or antibody-free control (lanes 6, 9, 12, and 15). The subsequently purified DNA from immunoprecipitated samples and unimmunoprecipitated samples (labeled as Input, lanes 5, 8, 11, and 14) was subjected to PCR amplification with primers specific for PKCδ promoter region that generates a 312-bp fragment. B, EMSA to test binding of nuclear proteins from NIE115 cells with the Sp site of the PKCδ promoter. EMSA was performed with an IRye700-labeled probe corresponding to the PKCδ promoter GC(1) and GC(2) motifs and 10 μg of nuclear extract from NIE115 cells. As indicated, various competitors (100-fold excess of unlabeled oligonucleotides, lanes 3–8) were added to the mixture before adding probe. The sequences of the competitors are shown in supplemental Table S2. The specific and nonspecific (labeled as N.S.) complexes are indicated by arrows.