Skip to main content
. 2016 Nov 17;6:37310. doi: 10.1038/srep37310

Figure 3. SPDEF directly binds to the promoter region of AIbZIP.

Figure 3

(a) Left: schematic representation of reporter constructs containing indicated promoter regions upstream of the human AIbZIP gene. Luc: luciferase reporter gene. Right: luciferase reporter analysis using HEK293T cells co-transfected with each reporter construct and a vector expressing FLAG-tagged SPDEF (mean ± s.d., n = 6; ***P < 0.001, vs. pGL3-basic reporter plasmid without a promoter region). (b) Left: WB analysis for the expression of truncated SPDEF mutants using anti-FLAG antibody. SPDEF: FLAG-tagged full-length SPDEF, ΔETS: FLAG-tagged SPDEF lacking the ETS domain, ΔSAM: FLAG-tagged SPDEF lacking the SAM pointed domain, ΔETSΔSAM: FLAG-tagged SPDEF lacking both the ETS and the SAM pointed domains. Middle: schematic representation for each SPDEF mutant construct. The SAM pointed domain and the ETS domain are indicated. Right: luciferase reporter analysis using HEK293T cells co-transfected with a reporter construct containing −1 kb upstream of AIbZIP and vectors expressing a series of truncated SPDEF mutants (mean ± s.d., n = 3; **P < 0.01, vs. SPDEF). (c) Schematic representation of −1 kb upstream of AIbZIP. Ten GGA(A/T) core consensus sequences (−940 to −937 bp, −874 to −871 bp, −867 to −864 bp, −645 to −642 bp, −607 to −604 bp, −434 to −431 bp, −308 to −305 bp, −169 to −166 bp, −66 to −63 bp, −40 to −37 bp), and the annealing sites of each primer set (P1-P4) used in ChIP assays are indicated. (d) LNCaP cells were infected with a vector expressing FLAG-tagged SPDEF for 72 h before immunoprecipitation of the chromatin with anti-FLAG or anti-Histone H3 antibodies. Mouse IgG was used as a negative control. The purified input DNAs and the immunoprecipitated DNAs were analyzed by PCR using the primer sets shown in (c). Full-length gels and blots are presented in Supplementary Figure S10.