Fig. 7. A complex formation of wild-type Wat1, but not mutant protein and
immunopurification of the Wat1-containing complex. (A,B) Gel filtration
chromatography. Soluble cell extracts were prepared from a wild type-tagged
(A, Wat1-HA) or wat1-5235 mutant-tagged strain (B, Wat1-5235-Myc) and
loaded onto Superose 6 columns. Each fraction together with total extracts (10
µg, shown as T) was run on an SDS-PAGE and immunoblotting was performed
with anti-HA (A) or anti-Myc antibody (B). Positions of size markers (2000
kDa, 669 kDa and 43 kDa) are also shown. (C) Autoradiogram of
immunoprecipitated proteins from a Wat1-HA strain with the anti-HA antibody is
shown. Cell extracts were prepared from a Wat1-HA (lane 1) or non-tagged
wild-type strain (lane2), which was metabolically labelled with
Tran[35S]-label. Protein bands that are specifically precipitated with anti-HA
antibody are marked by arrows, and the band corresponding to Wat1-HA is also
shown (identified with immunoblotting). The positions of molecular weight
markers are on the right.
