PPARγ activates the calreticulin gene. (A) A schematic representation of reporter plasmids used in this study. The calreticulin promoter (pLCC0) contains two PPRE sites: PPRE1 (−1,944 bp) and PPRE2 (−590 bp). Two promoter deletion constructs (pLC1 and pLC2) and two promoter constructs, one containing a mutation of PPRE1 (pLC0mt1) and the other containing a mutation of PPRE2 (pLC0mt2), were used to identify PPRE sites activated by PPARγ on the calreticulin promoter. (B) To analyze the effect of PPARγ and RXRα on the calreticulin promoter, NIH3T3 cells were transiently cotransfected with the PPARγ and RXRα expression plasmids with a luciferase reporter gene controlled by the calreticulin promoter as indicated in A. Individual controls were obtained for each expression plasmid, and data were plotted relative to that control (luciferase/β-galactosidase). The data shown are mean ± SD (error bars) of three independent experiments. (C) EMSA analysis was performed to demonstrate the binding of PPARγ to the PPRE1 and PPRE2 elements on calreticulin promoter. The positions of PPARγ–RXRα–DNA complexes are indicated. White lines indicate that intervening lanes have been spliced out. (D) Supershift EMSA analysis of PPARγ binding to the PPRE1 and PPRE2 elements on calreticulin promoter using anti-HA tag antibody was used to show the binding of transcriptional factor to the DNA element. Mutated PPRE1 and PPRE2 oligonuleotide show no interaction with PPARγ. Arrows mark PPARγ complexes with either PPRE1 or PPRE2. (E) ChIP analysis of a putative PPARγ-binding site in the mouse calreticulin promoter was used to show binding of PPARγ to the endogenous calreticulin promoter. Lane 1 shows input DNA, lane 2 is a negative control without antibody treatment, and lane 3 shows ChIP with antibodies.