Article Title: Quantitative proteomics links the LRRC59 interactome to mRNA translation on the ER membrane
Figure Lengend Snippet: Identification of ER membrane protein interactomes by proximity proteomics. (A) Schematic of known (SEC61 translocon, OST complex), and candidate (SEC62, LRRC59) ER-ribosome receptors. SEC61β (purple), a subunit of the SEC61 translocon, RPN1 (green), a subunit of the OST complex, SEC62 (orange), and LRRC59 (blue) are expressed as BioID chimeras, labeling interacting and near-neighbor proteins (indicated by starred ribosomes and proteins W, X, Y, and Z). (B) Left panel: Streptavidin blots examining the subcellular distribution of biotin-labeled proteins within HEK293 cells expressing either the LRRC59-, SEC62-, SEC61β-, or RPN1-BirA reporter constructs. Biotin labeling (doxycycline (dox)-inducible expression of reporters) was performed over a time course spanning 0-6 hours and cytosol (C) and membrane (M) extracts prepared by detergent fractionation. Right panel: Densitometric quantifications of biotin labeling intensities for cytosolic and membrane fractions. (C) Canine pancreas rough microsomes with (+BirA) or without (-BirA) the addition of BirA* in trans . Biotin labeling of proteins was conducted over 0-18 hours (top, left). Biotin labeling intensities were quantified using densitometric analyses (top, right). As a loading control, total protein lysate was analyzed by India ink staining (bottom, left) and quantified by densitometric analysis (bottom, right).
Article Snippet: Subcellular fractions were cleared by centrifugation (15,300 x g for 10 minutes).
Techniques: Labeling, Expressing, Construct, Fractionation, Staining