Lenses from 11-week or 11-month-old mice were dissected and their diameters were measured and used to calculate lens volume. Dissected lenses were homogenized in 0.1 M NaCl, 0.1 M Na
2HPO
4, 10 mM ascorbic acid, and protease inhibitors (10 μg/mL each of chymostatin, leupeptin, and pepstatin). After centrifugation at 14,000
g for 20 minutes at room temperature, the insoluble fraction was subjected to a second wash with 1 mL of 0.1 M NaCl, 0.1 M Na
2HPO
4, followed by a subsequent wash with 1 mL of 20 mM NaOH. The insoluble fraction was finally washed in 1 mL of 1 mM Na
2CO
3, after which the pellets were resuspended in a volume of sample buffer proportional to the total starting lens volume and stored at −80°C. Equal volumes of samples from each age were electrophoresed on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (GE Healthcare Bio-Sciences, Pittsburg, PA). Commercial protein standards (MagicMark; Invitrogen, Carlsbad, CA) were used as molecular weight markers. Blots were then probed with antibodies specific for the carboxy tail of Cx46 (rabbit
50 ), the cytoplasmic loop of Cx50 (goat; Santa Cruz Biotechnology, Santa Cruz, CA), or α-tubulin (mouse, provided by Dr I. Spector, Stony Brook University). Horseradish peroxidase-conjugated goat anti-rabbit, rabbit anti-goat, (Jackson ImmunoResearch, West Grove, PA), or sheep anti-mouse (GE Healthcare Bio-Sciences) secondary antibodies were used prior to enhanced chemiluminescence detection. Blots were visualized and band intensities were quantified using a commercial imager and software (FluorChem E and AlphaView; Protein Simple, Santa Clara, CA). Western blots of lenses from three mice each, at ages of 2 and 16 months, were compared (data not shown) and blots of lenses from three mice each, at ages of 11 weeks and 11 months were compared (see Results). Both studies showed reductions in labeling that were consistent with reductions in gap junction coupling conductance, but the 11-week versus 11-month blots had the advantage of using α-tubulin as a control for nonspecific reductions in labeling.