Research Paper Volume 13, Issue 13 pp 17568—17591

Aging-dependent loss of GAP junction proteins Cx46 and Cx50 in the fiber cells of human and mouse lenses accounts for the diminished coupling conductance

The automated western immunoblot (AWI) analyses of connexin 50 (Cx50) in normal and cataractous lenses of different age groups. AWI was performed on a Wes (ProteinSimple) as described recently [48, 56]. Briefly, each sample was loaded with 0.9 μg total protein and then analyzed with the Size Separation Master Kit and Split Buffer (12–230 kDa) according to the manufacturer’s standard instruction using anti-Cx50 antibody (for antibody information, see Experimental Procedures) with a dilution factor of 1:20. The Campass software (Protein Simple, version 4.1.5) was used to program the PeggySue-robot and for presentation (A) and quantification (B–C). Output western blot style data (A) were displayed with exposure time indicated in Figure 1, and the quantification data (B–C) were displayed from the software-calculated average of seven exposures (1–512s). (B) Quantification results show gender difference. Each bar represents an average of 8 samples for cataract lenses but one sample for normal human lens of 40s and three samples for normal human lenses of 60s. Lanes 1–2 represent normal lenses, and lanes 3–18 represent cataractous lenses of different age groups (3–6, 50s; 7–10, 60s; 11–14, 70s and 15–18, 80s). (C) Quantification results show age difference. *p

Figure 3. The automated western immunoblot (AWI) analyses of connexin 50 (Cx50) in normal and cataractous lenses of different age groups. AWI was performed on a Wes (ProteinSimple) as described recently [48, 56]. Briefly, each sample was loaded with 0.9 μg total protein and then analyzed with the Size Separation Master Kit and Split Buffer (12–230 kDa) according to the manufacturer’s standard instruction using anti-Cx50 antibody (for antibody information, see Experimental Procedures) with a dilution factor of 1:20. The Campass software (Protein Simple, version 4.1.5) was used to program the PeggySue-robot and for presentation (A) and quantification (BC). Output western blot style data (A) were displayed with exposure time indicated in Figure 1, and the quantification data (BC) were displayed from the software-calculated average of seven exposures (1–512s). (B) Quantification results show gender difference. Each bar represents an average of 8 samples for cataract lenses but one sample for normal human lens of 40s and three samples for normal human lenses of 60s. Lanes 1–2 represent normal lenses, and lanes 3–18 represent cataractous lenses of different age groups (3–6, 50s; 7–10, 60s; 11–14, 70s and 15–18, 80s). (C) Quantification results show age difference. *p < 0.05.