FIG. 1.

Regulation of GC-C activity by tyrosine phosphorylation in intestinal cells (A) Fluid accumulation was monitored in ligated ileal loop assays following injection of ST (100 nM) or 8-Br-cGMP (5 mM) in the presence or absence of HgCl₂ (50 μM) and PV (0.5 mM). The experiment was repeated twice with two loops per treatment, and the values shown represent the mean ± standard error of the mean (*, P < 0.05). Inset, pY Western blot with mucosal scrapings from rat intestinal loops with or without treatment with PV and HgCl₂. (B) Mucosal scrapings were prepared from treated and untreated intestinal loops as indicated, and solubilized proteins were allowed to interact with normal mouse immunoglobulin G (NMI) or GCC:B10 monoclonal antibody. Immunoprecipitates (IP) were subjected to Western blot analysis with monoclonal antibody GCC:4D7 and pY antibodies. (C) T84 cells were infected with lentiviruses encoding control or c-src shRNA. Subsequently cells were treated with HgCl₂ and PV and harvested, and Western blotting was performed with total c-src, phospho-c-src, and GCC:C8 antibodies. The data shown are representative of experiments repeated thrice. (D) Lysates were prepared from T84 cells infected with lentivirus encoding either control or c-src shRNA following treatment with PV and HgCl₂ or medium alone. Lysates were subjected to Western blotting with pY antibodies. The data shown are representative of experiments repeated thrice. (E) T84 cells were infected with lentivirus encoding control or c-src shRNA. Cells were then treated as indicated, following which ST (100 nM) was added and intracellular cGMP produced measured by radioimmunoassay. Values shown represent the mean ± standard error of the mean of duplicate determinations in experiments repeated thrice (*, P < 0.01).