Figure 5.

Coimmunoprecipitation of Lhs1p with Hsp150Δ–β-lactamase. (A and B) H647 (lhs1⁻ c-myc–LHS1 ⁺ sec18) cells were incubated for 1 h at 37°C (lanes 1 and 6), 20 min at 50°C (lanes 2 and 7), and at 24°C with CHX for 3 h (lanes 3 and 8) or 6 h (lanes 4 and 9). The samples were lysed under mild detergent conditions and divided in two. One set of samples was subjected under nondenaturing conditions to immunoprecipitation (IP) with anti– β-lactamase antiserum (α-bla; A and B, lanes 1–4) and the other with preimmune serum (pim; A and B, lanes 6–9). The precipitates were separated by SDS-PAGE, blotted, and immunostained (IS) using anti–c-myc antibody (α-c-myc; A, lanes 1–10) or anti– β-lactamase antiserum (B, lanes 1–10). Lanes 5 and 10 in both panels contained total lysate sample. c-myc–Lhs1p (lhs1p) and Hsp150Δ–β-lactamase (bla) are indicated by arrowheads. Molecular mass markers of 212, 170, 116, and 76 kD are indicated in lane M. (C) A cell sample incubated at 37° and 50°C, like above in lane 2, was lysed like above and centrifuged in a 10–40% sucrose gradient as described in Fig. 2. The fractions (lanes 1–10) and pellet material (lane P) were immunoprecipitated with anti– β-lactamase antiserum and subjected to blotting and immunostaining with anti–c-myc antibody, as in A. T, total cell lysate sample stained with anti–c-myc antibody. Arrowhead indicates c-myc–Lhs1p.