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. 1998 Jul;9(7):1661–1674. doi: 10.1091/mbc.9.7.1661

Figure 4.

Figure 4

(A) SCAMP3 is tyrosine phosphorylated in EGF-stimulated NeoR cells. Serum-starved NeoR cells were either unstimulated (lanes 1, 3, and 5) or stimulated (lanes 2, 4, and 6) with 100 ng/ml EGF for 30 min, lysed in CHAPS buffer, and immunoprecipitated with either anti-SCAMP (7C12, lanes 1 and 2), anti-SCAMP3 (3γ, lanes 3 and 4), or a control antibody (9E10, lanes 5 and 6). The immunoprecipitates were blotted for phosphotyrosine (αP-Tyr, upper panel) or for SCAMP3 (3γ, lower panel). (B) Reimmunoprecipitation of SCAMP3. NeoR cells were EGF stimulated for 30 min, lysed in CHAPS buffer, and immunoprecipitated with anti-SCAMP (7C12). Samples 2, 3, and 4 were boiled in 1% SDS and reconstituted in CHAPS buffer, and then reimmunoprecipitated with anti-SCAMP (7C12, lane 2), anti-SCAMP3 (3γ, lane 3), or a control antibody (9E10, lane 4). Samples were Western blotted for phosphotyrosine and visualized using ECL. (C) Reimmunoprecipitation of SCAMP1. NeoR cells were stimulated with EGF for 1 h, lysed in CHAPS buffer, and immunoprecipitated with anti-SCAMP (7C12, lanes 1–3) or anti-SCAMP1 (1Ω, lanes 4 and 5) in the first round. Samples 2 and 3 were boiled in SDS and reconstituted in buffer containing 1% CHAPS, and then subjected to a second round of immunoprecipitation with either anti-SCAMP1 (1Ω, lane 2) or a control antibody (anti-transferrin receptor, lane 3). Samples were resolved by SDS-PAGE, transferred to nitrocellulose, Western blotted with either anti-phosphotyrosine (lanes 1–4) or anti-SCAMP (lane 5), and visualized by ECL.