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.