Fig. 3.

Bat3 is required for Sec61β-OPG membrane integration. Samples of rabbit reticulocyte lysate were immunodepleted of Bat3 or TRC40, subjected to a parallel control immunodepletion using appropriate chicken or rabbit antibodies, or left completely untreated, as indicated. (A) Upper panel, the ability of different reticulocyte lysate preparations to support the membrane integration of Sec61β-OPG was assessed via N-glycosylation (+gly and −gly) following immunoblotting with the anti-opsin monoclonal antibody. Lower panels, reticulocyte lysate was left untreated or treated with increasing amounts of a chicken anti-Bat3 IgY or a control chicken IgY as indicated, and samples immunodepleted using a secondary antibody. The resulting levels of Bat3, TRC40, Hsp/Hsc70 and SRP54 were determined by immunoblotting as indicated. Individual boxes show scans from one exposure of a single piece of film. (B) Upper panel, the ability of different lysate preparations to support the membrane integration of Sec61β-OPG was determined as described in A. This panel shows the scan from a single piece of film with two irrelevant lanes separating the TRC40 depletion (lane 2) and the parallel control sample (lane 3) removed for simplicity, as indicated by the vertical line. Lower panel, the levels of TRC40 and Bat3 were determined as for A. (C) The ability of different lysate preparations to support the membrane integration of Cytb5-OPG was determined as for A. A small amount of SDS-resistant Cytb5-OPG dimer was observed. In some cases, samples subjected to immunodepletion contained residual immunoglobulin heavy chain (labelled IgG) that crossreacted with the secondary antibody (see A and B, TRC40 immunoblot, white dots). A minor species observed with the TRC40 serum is also indicated (B and C, TRC40 panel, filled square).