The amplified ISX cDNA product then was cloned into the pCDNA 3.1 V5/His TOPO vector by following the manufacturer��s instructions (Invitrogen) and transformed into selleck bio TOP10 cells according to the manufacturer��s instructions (Invitrogen). Positive clones were selected and subjected to plasmid DNA minipreparation according to manufacturer��s instructions (Qiagen, Valencia, CA, USA). Finally, appropriate construction of wild-type (WT) ISX ORF (pISX-WT) in the pCDNA 3.1 V5/His TOPO vector was verified by sequence analysis on both strands (Genomics Core Sequencing Facility, Case Western Reserve University, Cleveland, OH, USA). Indirect immunofluorosence and confocal microscopy For immunostaining experiments, CaCo-2 cells were seeded at a density of 2 �� 105 cells on coverslips in 6-well plates and allowed to adhere for 24 h.

Cells were treated with RA (1 ��M) or control vehicle (ethanol) and incubated for 12 h. Then cells were fixed in a freshly prepared mixture of 4% formalin in phosphate buffered saline (PBS; pH 7.4) for 20 min at room temperature. After multiple washes with PBS, cells were incubated with blocking buffer (2% BSA and 0.2% Triton-X 100 in PBS) for 15 min at room temperature. The primary polyclonal rabbit antiserum anti-ISX prepared at a 1:500 dilution in blocking buffer was then added to these cells and incubated for 1 h at room temperature. The primary antibody was removed, and cells were washed gently 3 times with PBS for a total of 30 min. The secondary antibody anti-rabbit conjugated Alexa 488 was diluted 1:500 in blocking buffer, added to the cells, and incubated for 1 h at room temperature in darkness.

After further washing in PBS, coverslips with cells were mounted facedown onto glass slides (Labtek, Scotts Valley, CA, USA) with a small drop of ProLong Gold antifade mounting medium containing DAPI (Molecular Probes). The next day, cells were examined at room temperature under a Zeiss LSM 510 UVMETA confocal microscope with an HCX Plan ��40 numerical aperture 1.4 oil-immersion objective lens (Zeiss, Jena, Germany). Images were acquired with Zeiss Anacetrapib confocal software version 2.0. All experiments were performed in triplicate, and ~100 cells/experiment were counted. ChIP assay and PCR CaCo-2 cells were cultured in 150-cm2 dishes and, on 90% confluence, were treated with 1% formaldehyde at 37��C with gentle swirling for 10 min to enable crosslinking of nuclear proteins with genomic DNA. Then the ChIP assay, to evaluate binding of RARs to the ISX promoter, was performed essentially as described by the manufacturer (Millipore). About 4 ��g of RAR antibody (M-454; Santa Cruz Biotechnologies) was used for immunoprecipitation, and IgG was used as the negative control.