Figure 4: TOX3 binding to a GAS sequence accessed by an Immobilized DNA-binding assay.
A) T24 bladder cancer cells were transiently transfected with an empty vector pcDN3.1 V5-His (mock) or the pcDN3.1 TOX3/V5-His vector (TOX3) was incubated with a STAT1 promoter fragment (438 bp). Immunoprecipitation was achieved by either anti-V5 (lanes 1, 3 and 5) or IgG antibodies (lanes 2,4 and 6). A) 1 μl eluted bound DNA analyzed on an Agilent DNA chip. MW: molecular weight marker (bp size); lane 1,2: cell extracts over expressing TOX3; lane 3,4: cell extracts transfected with a mock, lanes 5, 6: without cell extract; lane 7 cell extracts over expressing TOX3 but without antibody; lane 8: extracts from mock treated cells without antibody; lane 9: purified DNA fragment, no cell extract, no antibody.
B) RT-qPCR analysis of the samples described in Fig 4A calculated the amount [fg] of DNA retrieved. TOX3 bound to the DNA with the GAS element and in the presence of TOX3 we were able to immunoprecipitate 542 fg DNA/μl in the presence of the anti-V5 antibody (black), compared to 125 fg DNA/μl with the IgG antibody (white). The control reactions retrieved negligible amounts of DNA.