SCAMP3 is tyrosine phosphorylated in
vanadate-treated CHO cells. (A) CHO cells were either untreated (lanes
1, 3, and 6) or treated (lanes 2, 4, and 7) with 5 μM pervanadate for
20 min and immunoprecipitated with anti-SCAMP (7C12, lanes 3–5),
anti-SCAMP3 (3γ, lanes 6–8), or a control antibody (9E10, lanes 1
and 2). Two samples (lanes 5 and 8, +/−) were washed in vanadate-free
media and further incubated for 1 h. Immunoprecipitates were
Western blotted with anti-phosphotyrosine (αP-Tyr, upper panel) or
anti-SCAMP3 (3γ, lower panel) and visualized using ECL. The
corresponding 7C12 blot demonstrated that bands with the mobility of
SCAMP1 and SCAMP3, but not SCAMP2, were phosphorylated on tyrosine (our
unpublished observations). (B) Untreated (lanes 1, 3, and 5) or
vanadate-treated (lanes 2 and 4) CHO cells were lysed in CHAPS buffer
and immunoprecipitated with anti-SCAMP1 antibodies 1α (lanes 1, 2,
and 5) or 1ς (lanes 3 and 4). Samples were resolved by SDS-PAGE,
transferred to nitrocellulose, and Western blotted with
anti-phosphotyrosine antibody (αP-Tyr, lanes 1–4) or with anti-SCAMP
(7C12, lane 5). The positions of SCAMPs 1, 2, and 3 are indicated (Sc1,
Sc2, Sc3), as well as the position of several unidentified
phosphotyrosine proteins (lanes 2 and 4, arrowheads) and the IgG heavy
chain (HC). (C) Tyrosine phosphorylation of endogenous SCAMP3 and
transiently overexpressed SCAMP3-myc. 293T cells were transfected with
expression vector pBK alone (lanes 1 and 2) or with human
SCAMP3-myc/pBK (lanes 3 and 4), lysed in CHAPS buffer 48 h
post-transfection, and immunoprecipitated with anti-myc antibody (9E10,
lanes 1–4). The supernatants from samples 1–4 were then
immunoprecipitated with anti-SCAMP antibody (7C12, lanes 5–8).
Immunoprecipitates were Western blotted with anti-phosphotyrosine
antibody (upper panels) or anti-myc (lower panel) and visualized by
ECL. An unknown protein of ∼40 kDa coimmunoprecipitates with
SCAMP3-myc (lanes 4 and 8, arrowhead).