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. 2011 Dec 7;40(7):3006–3017. doi: 10.1093/nar/gkr1197

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© The Author(s) 2011. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Independent association capacity of p180 with the translocon and ribosomes. (A) After removal of the cytosolic fractions with the permeabilization and wash buffers, the membrane fractions of ascorbate-treated cells were either left untreated or stripped of ribosomes by in situ EDTA treatment. Subsequently, membrane fractions were prepared with lysis buffer containing digitonin. The control (untreated) or ribosome-stripped membrane fractions were analyzed on a 5–20% sucrose gradient at 0.4 M KOAc. Marker proteins on western immunoblots and rRNAs in agarose gels analyzed for control membranes (left panel) and ribosome-stripped membranes (right panel) are shown. Flow schema for preparing the ribosome-stripped and untreated membranes are shown in Supplementary Figure S4. (B) Ribosome-stripped membrane fractions were prepared as described for (A) and subsequently centrifuged through a 0.5 M sucrose cushion to remove residual ribosomes. The resulting ribosome-free membrane fractions were subjected to IP analysis with control IgG (lane 2), an anti-p180 antibody (lane 3) and an anti-Sec61β antibody (lane 4) in the presence of 0.4 M KOAc. In the lanes containing the IP samples 10 times higher amounts were loaded compared with the input samples (lane 1). Flow schema for preparing the ribosome-free membranes are shown in Supplementary Figure S4. (C) Ribosome-stripped membrane fractions prepared with 1% NP-40 lysis buffer were further treated with 0.5% deoxycholate and 0.1% SDS at 4°C for 1 h to dissociate possible complexes in the presence of 0.4 M KOAc. After sedimentation at 100 000g for 40 min through a 0.5 M sucrose cushion, the supernatants were subjected to the IP assay with an anti-p180 antibody to prepare p180-beads. Marker proteins were analyzed for the input sample (lane 1), p180-beads (lane 2) and control-beads (lane 3) to confirm the loss of the translocon-related proteins. (D) The p180-beads or control-beads were incubated with isolated monosomes or the subunits at 4°C for 1 h at 0.4 M KOAc. After careful washing, the tested beads were recovered by a magnet and analyzed for rRNA by agarose electrophoresis and for p180 by western blotting. The monosome, 60S and 40S preparations contained no detectable levels of Sec61β, ribophorin II, TRAPα or p180 (data not shown). Lanes 1, 5 and 9: input p180-beads; lane 2: input 80S ribosomes; lanes 6 and 10: input ribosome subunits; lanes 3, 4, 7, 8, 11 and 12: recovered bead fractions after incubation.