After 18 hours of com pound treatment, the cells were washed with

After 18 hours of com pound treatment, the cells were washed with 50 uL 1�� PBS and lysed with 50 uL Tropix lysis buffer. Cell lysate was mixed 1 1 with luciferase substrate, and luminescence was measured with a 700 nm filter on a Victor X5 microplate reader. The Bradford method was used to measure total protein in each sample. Raw luciferase data was normalized to both total protein http://www.selleckchem.com/products/jq1.html and background luciferase Inhibitors,Modulators,Libraries expression in the DMSO control samples and expressed as fold increase over DMSO. Inducible knockdown of AhR by lentiviral infection pSUPER vectors were constructed using two previously published siRNA sequences directed toward the AhR, by standard cloning procedures. The siRNA cassette downstream of the H1 pro moter was sequenced to confirm accuracy, excised from pSUPER, and subcloned into the lentiviral vector pLVTHM.

Viral particles containing shAhR vectors were created by transfecting host 293 T cells with vectors en coding for VSVG, a lentiviral vector coat protein, PAX2, a packaging plasmid, and pLVTHM Inhibitors,Modulators,Libraries shAhR using stand ard protocols. Briefly, subconfluent 293 T cells were transfected with 0. 5 ug VSVG, 1 ug PAX2, and 1. 5 ug pLVTHM shAhR using Trans IT LT1 transfection reagent. After six hours, medium was changed and recombinant lentivirus vectors were harvested 24 hours later. Using a similar protocol, pLV tTR KRAB recombinant lentivirus was produced. pLV tTR KRAB encodes a tetracycline controlled hybrid protein con taining the Tet repressor and the Kr��ppel associated box domain of human Kox1. The purpose of KRAB in Tet responsive systems is described elsewhere.

MDA MB 468 and Cal51 cells were seeded subconflu ently in a six well tissue culture plate at 37 C and 5% CO2. Twenty four hours later, media were removed and replaced with 1 mL of DMEM supplemented with 10% FBS contain ing recombinant pLV tTR KRAB and 5 ug/mL polybrene. After allowing Inhibitors,Modulators,Libraries two passages for recovery, the MDA MB 468 and Cal51 cells were subjected to the same protocol, substituting pLV tTR KRAB with the two pLVTHM shAhR lentiviruses, producing MDA MB 468shAhR and Cal51shAhR cell lines. Western blot analysis MDA MD 468shAhR and Cal51shAhR were treated for seven days with vehicle or 750 ng/mL doxycycline in DMEM with 10% Tet Approved FBS. After treatment, cells were collected by trypsinization, washed with 1�� PBS, and lysed using Triton X 100 lysis buffer.

Total protein concentration was measured using the Bradford Inhibitors,Modulators,Libraries method, and 20 ug of protein was resolved using SDS PAGE on 8% polyacrylamide gels. Protein was transferred to a nitrocellulose membrane at 4 C for one hour at 0. 35A. Inhibitors,Modulators,Libraries Membranes were blocked with 5% nonfat sellectchem milk in PBS 0. 1% Tween for one hour at room temperature, then incubated with 1 10,000 anti AhR antibody or 1 10,000 anti B Actin overnight at 4 C. Membranes were incu bated with 1 10,000 goat anti rabbit HRP or anti mouse HRP secondary antibody for one hour at room temperature.

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