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

Age-dependent changes of the connexins Cx43, Cx46 and Cx50 in mouse lens epithelial samples of different age groups as determined by the automated western immunoblot (AWI) analyses. 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 indicated antibodies (for antibody information, see Experimental Procedures) with a dilution factor of 1:100 for Cx43, and 1:20 for Cx46 and Cx50. The Campass software (Protein Simple, version 4.1.5) was used to program the PeggySue-robot and for presentation (A) and quantification (B–D). Output western blot style data (A) were displayed with the best exposure determined by the software, and the quantification data (B–D) were displayed from the software-calculated average of seven exposures (1–512s). (B) Quantification results show age difference of Cx43. (C) Quantification results show age difference of Cx46. (D) Quantification results show age difference of Cx50. NS, not significant, *p **p

Figure 6. Age-dependent changes of the connexins Cx43, Cx46 and Cx50 in mouse lens epithelial samples of different age groups as determined by the automated western immunoblot (AWI) analyses. 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 indicated antibodies (for antibody information, see Experimental Procedures) with a dilution factor of 1:100 for Cx43, and 1:20 for Cx46 and Cx50. The Campass software (Protein Simple, version 4.1.5) was used to program the PeggySue-robot and for presentation (A) and quantification (BD). Output western blot style data (A) were displayed with the best exposure determined by the software, and the quantification data (BD) were displayed from the software-calculated average of seven exposures (1–512s). (B) Quantification results show age difference of Cx43. (C) Quantification results show age difference of Cx46. (D) Quantification results show age difference of Cx50. NS, not significant, *p < 0.05, **p < 0.01.