Figure 1. Interaction between GST-AQP0-C and filensin in rat lens extract. A: The interaction between purified GST-AQP0-C and lens filensin. Western blotting analysis was performed with anti-filensin outer tail domain antibody. Lane 1: urea fraction of rat lens extract showing 94 kDa filensin. Purified GST-AQP0-C incubated with lens extract (lane 2). Filensin (94 kDa) was detected by anti anti-filensin outer tail domain antibody that interacts with the C-terminal domain of AQP0. Lane 3 contained purified GST-AQP0-C as a control. B: The interaction between lens filensin and purified GST-AQP0-C or its pseudophosphorylated forms (1, 3, or 5 sites mutated in the COOH-terminal peptide) was investigated by western blotting with anti-filensin rod antibody. Lane 1: urea fraction of rat lens extract showing 94, 50, and 38 kDa filensin protein bands. Purified GST-AQP0-C or GST-AQP0-C with 1, 3, or 5 pseudophosphorylated sites interacts with the 94 and 50 kDa filensin bands but not with the 38 kDa band (lanes 2, 4, 6, and 8, respectively). In the absence of lens extract, neither the purified GST-AQP0-C nor the 1, 3, or 5 site-mutated AQP0-C constructs interacted with the anti-filensin rod antibody (lanes 3, 5, 7, and 9, respectively). As a further control, when GST alone was incubated with lens extract, it did not interact with lens filensin (lane 10). Lane M in A and B shows the molecular markers.