Figure 10.

Alteration of CES regulation is concurrent with the disappearance of the LSU₈-RAF1 oligomer. (A) Immunoblot with the Rubisco antibody after CN-PAGE analysis of soluble protein fractions of WT (note the dilution), ΔrbcL, ΔRBCS;LSU₈mut, and ΔRBCS;LSU₈mut strains. The position of the LSU-complexes observed in ΔRBCS is indicated at the right using the same symbols as in Figure 5 (square, cross, and circle). The empty triangle and dashed box indicate the somewhat diffuse band attributed to LSU dimer. (B) Immunoblot after CN-PAGE analysis (4–16%) of soluble protein fractions from ΔRBCS (top) and ΔRBCS;LSU₈mut (bottom), followed by a second dimension run on a 13% SDS-PAGE gel using the anti-Rubisco and anti-RAF1 antibodies sequentially. The Rubisco antibody was stripped before rehybridization with the anti-RAF1 antibody, however a cross-reacting signal labeled with a red cross could not be completely stripped off. The position of the LSU-complexes observed in ΔRBCS and ΔRBCS;LSU₈mut are indicated on top of the gels using the same symbols as in A (square: LSU-CPN60, cross: LSU₈-RAF1 and circle: LSU₂-RAF1). The empty triangle denotes the band observed in RBCS;LSU₈mut attributed to RAF1-free LSU dimers. No corresponding signal can be detected at this position (dashed rectangle) with the RAF1 antibody in the ΔRBCS strain.