Figure 2.

Immunoprecipitation of Fus3-HA from ³⁵S-labeled cells. Strains EY960 and EY1124 (EY940 with FUS3 [pYEE114] or FUS3-HA [pYEE121]) was labeled with ³⁵S (see MATERIALS AND METHODS), and cells were harvested either without inducing with α factor, or after 20 and 60 min of α factor induction. Whole-cell extracts were prepared as described (see MATERIALS AND METHODS) and 200 μg of total protein was immunoprecipitated with 12CA5 for analysis. (A) Effect of salt in the immunoprecipitation. Samples were separated on a 10% (38:3 acrylamide:bis-acrylamide) SDS gel. Shown is an 18-h exposure of the autoradiogram. (B) Effect of detergents in the immunoprecipitation. Samples were separated on a 7.5% (30:0.8 acrylamide:bis-acrylamide) SDS gel. Shown is a 90-h exposure of the autoradiogram. For both panels A and B, extracts were prepared as described in MATERIALS AND METHODS and were then immunoprecipitated under the conditions described in the figure (note that in panel B, Set 1 also contained 1% bovine serum albumin and Set 2 also contained 1% ovalbumin). After immunoprecipitation, samples were washed with modified H-buffer (Elion et al., 1993). Fus3-HA is indicated by the arrow. ±HA indicates whether the strain contains Fus3-HA (+HA) or Fus3 (−HA). The dots indicate the positions of the 60- and 70-kDa proteins in panel B. DOC is deoxycholate. Extracts in panels A and B are from separate experiments.