IMAGE

Fig. 2

ID
ZDB-IMAGE-200406-17
Source
Figures for Zhang et al., 2020
Image
Figure Caption

Fig. 2 BRAWNIN (BR) is a conserved SEP at the inner mitochondrial membrane.

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.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Nat. Commun.