To characterize the distribution of the protein and activity levels of various MAPKs and the upstream activating kinases for ERKs in human, bovine, and rat lenses, we developed a method to isolate various fractions of fiber cells from the cortical to the nuclear layer after removal of the lens capsular epithelium.
Figure 1 depicts the capsular epithelium and different fractions of fiber cells that were isolated as described in the Methods section. After removal of the lens epithelium or each fraction of fiber cells, the remaining fiber mass was measured in both weight and size. The results are shown in
Table 1 . To further characterize different fractions, we examined the levels of αA- and αB-crystallin, and then compared them with the distribution of PKCα and GAPDH in the isolated lens epithelium and the different layers of fiber cells
(Fig. 2) . Although the weight of each fraction of fiber cells varied in human, bovine, and rat lenses
(Table 1) , the expression patterns of αA- and αB-crystallins in these lenses were common: both crystallins became gradually decreased from the cortical (F1) layer of fiber cells toward the N fiber cells (
Fig. 2A , 2–3, 6–7, 10–11; in citations of
Figures 2 3 4 5 6 7 8 , the numbers after the figure number correspond to the numbers to the right of the Western blots in panel A). Quantitative analysis of the two crystallins in each fraction of the fiber cells with an automated digitizing system revealed that the F1 fiber cells of the lenses from three different sources all contained approximately 40% to 50% of the total fiber cell αΑ- or αB-crystallin. In contrast, less than 2% of the total fiber cell αΑ- or αB-crystallin was found in the N fiber cells of these lenses
(Fig. 2B) . The expression pattern of GAPDH was also similar in the lenses of three different sources. Higher level of GAPDH was found in their lens epithelia and relatively lower level of GAPDH was found in their F1 fiber cells
(Fig. 2) . GAPDH was hardly detectable in F3 to N fiber cells
(Fig. 2) . Compared with αA- and αB-crystallin, and GAPDH, PKCα was differentially expressed in the fiber cells of human, bovine, and rat lenses. In human lens, PKCα was absent in the lens fiber cells (
Figs. 2A , 1;
2B , top). In the fiber cells of bovine lens, PKCα was detected only in the F1 fiber cells (
Figs. 2A , 5;
2B , middle). In contrast, PKCα was expressed in all fractions of the fiber cells except for the nucleus of 4-week-old rat lens (
Fig. 2A , 9). The PKCα level was gradually decreased from the epithelial cells to F3 fiber cells and became barely detectable in F4 fiber cells (
Fig. 2B , bottom). Although it was difficult to find a unique marker for each fraction of the isolated fiber cells, our determination of the weight, size, and relative expression of αA- and αB-crystallins in these fractions provided necessary characterization for each fraction of fiber cells.