Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Mar 11;11:1312. doi: 10.1038/s41467-020-14999-2

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 2 — a Ribosome protected fragment (RPF) reads from the BRAWNIN gene (C12orf73) in human cell lines. P1 and P2 refer to the 2 potential ORF isoforms generated by alternative splicing. b BR (P1) peptide is conserved throughout vertebrate evolution and possesses an N-terminal hydrophobic region that is predicted to be a transmembrane domain or signal peptide. Underlined regions are peptides detected by mass spec. Regions used as immunogens for raising the polyclonal antibodies used in this study are indicated. c Endogenous BR is detected in HeLa and HEK293T with an α-BR antibody. OE = overexpression. d Western blot of HEK293T lysates transfected with control or BR-targeting siRNAs. e Immunofluorescence of endogenous BR detected by α-BR in HeLa transfected with control or BR-targeting siRNAs. Scale = 5 μM. f Western blot of indicated proteins from subcellular fractions of HEK293T. W = whole cell, M = mitochondria enriched fraction, C = cytosol, P = nuclear fraction and debris, including un-extracted mitochondria; BR^∆N25 = synthetic BR peptide lacking the N-terminal 25 a.a. g Endogenous BR detected by α-BR in HeLa costained with mitotracker. Scale = 5 μM. h Endogenous BR detected by α-BR in HEK293T costained with mitotracker. Scale = 5 μM. i α-BR and α-citrate synthase (CS) immunofluorescence in mouse skeletal muscle, longitudinal section. Scale = 20 μM. j α-BR immunofluorescence and GFP epifluorescence of Mito-Dendra2 in mouse skeletal muscle, longitudinal section. Scale = 20 μM. k Extraction of endogenous BR from HEK293T mitochondria with buffer alone, Na₂CO₃ solution or buffer containing 1% Triton X. S = supernatant; P = pellet. l Extraction of endogenous BR from HEK293T mitochondria using increasing concentrations of digitonin. TOMM20, TOMM70, and VDAC1 are outer mitochondrial membrane (OMM) proteins, are MTCO1, ATP5A, and TIM23 are IMM associated. m Proteinase K protection assay of HEK293T mitochondria. The α-BR epitope is C-terminal to the predicted transmembrane domain. TOMM20 and TOMM70 have cytosolic domains. TIM23 has IMS domains. Both CS and ATP5A are in the matrix.