Figure 5.

14-3-3 Forms a Heterooligomeric Complex with preSSU and Hsp70.

(A) Wheat germ lysate–synthesized ³⁵S-preSSU (filled diamonds) and the nonphosphorylatable mutant ³⁵S-preSSU–M31/34-S/A (open diamonds) were fractionated in independent runs as before (see Figure 4) but with a fraction size of 250 μL. Only fractions of the relevant molecular size were collected. The positions of the molecular mass markers are indicated on top (open circles). Each fraction was precipitated by trichloroacetic acid, and precipitated proteins were separated by SDS-PAGE and subsequently transferred onto nitrocellulose membranes. Autoradiograms are shown below. Radioactivity present in each fraction was quantified by liquid scintillation counting after solubilizing nitrocellulose strips in dimethyl sulfoxide.

(B) Wheat germ lysate (∼130 μg protein) was used for coimmunoprecipitation by anti–14-3-3 and anti-Hsp70 antibodies. The immunoprecipitated polypeptides were separated by SDS-PAGE, blotted, and immunodecorated using 14-3-3 and Hsp70 antisera, as indicated by arrowheads. PIS, control experiment using preimmune serum.