Stable Ribosome Binding to the Endoplasmic Reticulum Enables Compartment-specific Regulation of mRNA Translation -- Stephens et al. 16 (12): 5819 Data Supplement - Supplemental Material -- Molecular Biology of the Cell

Stable Ribosome Binding to the Endoplasmic Reticulum Enables Compartment-specific Regulation of mRNA Translation
Mol. Biol. Cell Stephens et al. 16: 5819

Supplemental Material

This article contains the following supporting material:

Figure S1 - Generation of Cytosol and ER fractions.
(A-C)
In situ immunofluorescence of control (A) and detergent extracted (B, C) HEK293 cells; tubulin (red), TRAPα (green), and DAPI (blue). Cells were permeabilized by addition of 80ug/mL digitonin (B) and further solubilized with 2% dodecylmaltoside (C). Cells were then fixed by addition of paraformaldehyde and processed for immunofluorescent microscopy. Immunoblot analysis of cytosol and ER fractions generated from sequential detergent extraction or isopycnic flotation (c=cytosol, ER = endoplasmic reticulum, RM=rough microsome) for the resident ER membrane protein TRAPƒÑ and the cytosolic protein tubulin (D). NIH 3T3 cells were radiolabeled by addition of [³⁵S]-methionine/cysteine. Cytosol and ER fractions were subjected to indirect immunoprecipitation of tubulin or GRP94 and protein distribution determined by SDS-PAGE and phosphorimager analysis (E).
Figure S2 - mRNA distribution between the cytosol and ER compartments is maintained during the UPR
For the experiments shown in Fig.5, the mRNA distribution of specific messages between cytosol and ER fractions from control- and thapsigargin-treated NIH 3T3 cells using quantitative RNA dot blots. RNA samples were transferred to nylon membranes by vacuum filtration on a dot-blot apparatus and quantified following hybridization, washing and phosphorimager analysis.
Figure S3 - ER-Bound Ribosomes Preferentially Participate in mRNA Translation during the UPR
Cytosol (A, B), total membrane-derived ER (C, D) and rough microsome-derived (E, F) fractions from control (A, C, E) and DTT-treated (B, D, F) J558L cells were centrifuged through 15-50% linear sucrose gradients for 3 hrs at 151,000xg. The downward facing arrow denotes the 80S monosome. The data was quantified as the percentage of the total probe signal for a given mRNA, per gradient. BiP mRNA is represented by the solid line with open circles; ATF4 mRNA by the small dotted line with open squares; XBP-1 mRNA by the dashed line with open diamonds; and GAPDH mRNA by the large dotted line with Xs.