Time- and attachment-dependent site-specific phosphorylation of c-Src in ARPKD by comparison to normal HFCT cells. A: Western immunoblot analysis of site-specific phosphorylation of c-Src. Equal numbers of cells plated onto type I collagen for 30 min, 4 h, or 7–11 days (confluent steady state) and attached cells analyzed for pY418-Src and pY529-Src. Cell lysate, 30 μg per lane. Anti-pY418-Src, 1:1,000; anti-pY529-Src, 1:1,000. After initial immunoblotting, membranes were stripped and reimmunoblotted for total c-Src. Densitometry analysis was carried out and the fraction of phosphorylated c-Src to total c-Src was calculated. The experiment was repeated 3 times and showed similar results. B: immunohistochemistry of anti-pY418-Src in normal and ARPKD tissue sections. A: 16-wk normal human fetal kidney. B: 19-wk fetal early-stage ARPKD. C: 4-yr normal human kidney. D: 4-yr end-stage ARPKD. Aminoethylcarbazole (red) reaction product is seen at the apical membranes of the developing ureteric bud epithelium in normal fetal kidneys (A, arrowhead and inset) and in small cysts of ARPKD kidneys (D, arrows and inset). No staining is seen in normal pediatric kidneys. Nomarski optics. Scale bar represents 100 μm.