Figure 9.

In Vivo and in VitroBinding of Nuclear SREBP-1c and SREBP-2 to Rat NIS 5′-UTR A and B, In vitro binding of rat nuclear SREBP-1c (A) and rat nuclear SREBP-2 (B) to the SRE+59 of rat NIS 5′-UTR. EMSA was performed using in vitro-translated rat nuclear SREBP-1c or rat nuclear SREBP-2 and DIG-labeled oligonucleotide corresponding to either wild-type or mutated SRE+59. Fold molar excess of unlabeled specific probe for competition (human LDLR-SRE) is indicated. The use of DIG-labeled specific probe (human LDLR-SRE) and nonspecific probe (mutated human LDLR-SRE) is also indicated. EMSA is representative for 1 of 2 independent experimentations, each providing similar results. C and D, Chromatin immunoprecipitation of SRE+59 of rat NIS 5′-UTR using antibodies against rat SREBP-1 and SREBP-2. The gene sequence spanning the SRE+59 of rat NIS 5′-UTR and a random control sequence were analyzed by conventional PCR (C) and qPCR (D) in the immunoprecipitated chromatin of FRTL-5 cells. FRTL-5 cells were grown in 6H medium in 150-mm dishes until 70%–80% confluent, then switched to 5H medium (without TSH) for 5 days, and subsequently treated with 25-HC (5 μmol/L) in the absence or presence of TSH (10 U/L) for 24 hours. Rabbit IgG was used as control. The image from agarose gel electrophoresis is representative for 1 of 3 independent ChIP experiments, each providing similar results. Data from qPCR analysis represent means ± SD for the 3 independent experiments. M, DNA fragment size marker.