The cells were rinsed in Opti-MEM (Ambion, Inc.) and transfected with si RNA (100 n together using si PORT (Ambion, Inc.) according to the manufacturer’s directions.Additional sets of CV-1 cells were transfected with a scrambled si RNA to serve as a negative control.
Recently, Hoffman (SC7312) and α-tubulin (E9) were purchased from Santa Cruz Biotechnology, Inc. Normal mouse Ig G and horseradish peroxidase-conjugated goat antirabbit Ig were purchased from Dako Cytomation (Carpinteria, CA).
Monoclonal antibodies to α, and scrambled negative control small interfering RNA (si RNAs) were all purchased from Ambion, Inc. The African green monkey fibroblast cell line, CV-1 (American Type Culture Collection, Manassas, VA), which lacks the nuclear receptor for thyroid hormone, was plated at 5000 cells/cm.
The assay was performed following the basic-native gel protocol from the Protein Purification Facility (il/∼purification/Protocols/PAGE_Basic.html).
All test compounds were diluted to their final concentration in 0.04 Tris-HCl (p H 6.8), and bromophenol blue] and run out on a 5% basic-native gel for 24 h at 45 m A in the cold.
The medium was changed every 3 d, and the cell lines were passaged at 80% confluence.