Whole-cell lysates were prepared in either HEGDM buffer (10 mM HEPES, 1 mM EDTA, 0.1% NP40, 10 mM Na molybdate, and 250 mM NaCl with protease inhibitors [Mini Complete Tablet; Roche Applied Science, Indianapolis, IN], for HLE B-3, αTN4, and NIH-3T3 cells and freshly isolated human lens epithelia, or lysis buffer (50 mM Tris, 300 mM NaCl, 0.5% Triton X-100 with protease inhibitors [Mini Complete Tablet; Roche Applied Sciences], for HeLa cells). Cells were scraped into PBS, centrifuged at 1000 rpm for 5 minutes, resuspended in buffer, and incubated on ice for 10 minutes. The sample was then centrifuged at 17,000g for 10 minutes at 4°C, and the supernatant was transferred to a new tube. Freshly isolated human lens epithelia were homogenized by 12 strokes of a homogenizer (Dounce; Kontes, Vineland, NJ). The homogenate was incubated on ice for 5 minutes and centrifuged at 14,000 rpm in a microfuge for 5 minutes at 4°C. Protein concentrations were measured using the Bradford method (Bio-Rad, Hercules, CA). HeLa nuclear extract was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A 1:1000 dilution of a partially purified GR (RP-500; Affinity Bioreagents, Golden, CO); 1 μg of HeLa, HeLa nuclear, NIH-3T3, HLE B-3, and αTN4 extract; and 60 μg human lens epithelia extract were electrophorectically separated on 7.5% denaturing Tris-HCl gels (Bio-Rad), transferred to nitrocellulose membranes (Bio-Rad), blotted with the GR-specific primary antibody H-300 and goat anti rabbit secondary antibody (both from Santa Cruz Biotechnology). Detection was preformed with enhanced chemiluminescence (NEN Life Sciences, Boston, MA).