The 40-kDa tyrosine-phosphorylated polypeptide is
SCAMP3. (A) IgA protease cleaves SCAMP3. NeoR cells were either
unstimulated (lanes 1, 2, 5, and 6) or stimulated (lanes 3, 4, 7, and
8) with 100 ng/ml EGF for 30 min, lysed in CHAPS buffer, and
immunoprecipitated with 7C12. Samples were either incubated in PBS
alone (lanes 1, 3, 5, and 7) or in PBS plus 400 nM IgA protease (lanes
2, 4, 6, and 8) for 1 h. Samples were Western blotted for
phosphotyrosine (αP-Tyr, left panel) or for SCAMP3 (3γ, right
panel). “Sc3Δ” indicates the presence of the putative SCAMP3
cleavage fragment. (B) Transferrin receptor is not cleaved by IgA
protease. NeoR cells were stimulated with EGF for 30 min, lysed in
CHAPS buffer, and immunoprecipitated with anti-transferrin receptor
antibody (H68.4). Samples were incubated in PBS alone (lane 1) or in
PBS plus 400 nM IgA protease (lane 2) for 1 h. Immunoprecipitates
were Western blotted for transferrin receptor and visualized using
ECL.