Figure 5

NFI-A binds to the L1 gene in vivo. A, ChIP analysis of N2A cells expressing Myc-NFI-A. L1, NFI-A-DNA complexes in the region of the L1 gene containing the NFI full binding site were assayed by PCR using primers indicated in Fig. 1A. Lanes: NFI-A bs, precipitation from Myc-NFI-A bs-transfected cells; NFI-A st, precipitation from Myc-NFI-A st-transfected cells; mock, precipitation from mock-transfected cells; pos, positive control (mouse genomic DNA as PCR template); water, water control (without PCR template). Chst8 and Chst11, To further confirm the specificity of ChIP analysis, ChIP samples were analyzed by PCR using primers amplifying parts of the mouse Chst8 and Chst11 genes, respectively. Note the absence of PCR signals for Chst8 and Chst11 in the ChIP sample lanes. B, Expression of Myc-tagged NFI-A isoforms in N2A cells. N2A cells were transfected with 4.8 μg of NFI-A cDNA expression vectors to express Myc-NFI-A st or Myc-NFI-A bs, respectively, or with 4.8 μg pCMX plasmid ("mock"). 48 h after transfection, cell were lysed and whole cell extracts were analyzed on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with anti-Myc antibody for detection of recombinant Myc-NFI-A and with anti-GAPDH antibody to check for loading of comparable protein amounts.