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. Author manuscript; available in PMC: 2016 Apr 16.
Published in final edited form as: Mol Cell. 2015 Mar 19;58(2):269–283. doi: 10.1016/j.molcel.2015.02.018

Figure 2. Nascent Chain Translocation Stabilizes Higher-Order RAMP Complexes.

Figure 2

(A) SDS-PAGE of translations ± CRMs at indicated pPL truncations. tRNA was removed with RNase. (B) BN- and SDS-PAGE of RAMPs, corresponding to translations (+CRMs) in panel A. Major complexes A–E are indicated. (C) Mean percent of RAMPs recovered in complexes A and B or C at specified chain lengths ± SEM (n=6). (D) SDS-PAGE of truncated single-cysteine pPL mutants (Cys34, Cys56, Cys88) expressed in CRMs and treated with 0.5M NaCl or digitonin (dig) prior to pegylation (PEG-mal). “*” and “←” indicate unmodified and pegylated peptidyl-tRNA respectively. Schematic diagram illustrates effects of salt and digitonin on assembled RTC intermediates. Mean percent pegylation is shown ± SEM (n ≥ 3). (E) BN-PAGE of pPL-137 RAMPs (top) aligned with corresponding 2D-BN/SDS-PAGE (bottom) labeled with complexes A–E. Silver stains, color; autoradiograms, grayscale. See also Figure S2.