Journal: bioRxiv
Article Title: Sequence grammar and dynamics of subcellular translation revealed by APEX-Ribo-Seq
doi: 10.1101/2025.05.26.656194
Figure Lengend Snippet: (A) Schematics of constructs for APEX2 fusion protein expression. (B) Immunofluorescence analysis of the indicated proteins (detected by EGFP) and desthiobiotinylated proteins/RNAs (detected with Alexa Fluor 555-conjugated streptavidin). DAPI (4’,6-diamidino-2-phenylindole) was used to stain the DNA. The scale bar represents 10 μm. (C) Western blotting and RNA dot blotting for proteins and RNAs under the indicated conditions. Desthiobiotinylated proteins and RNAs were detected using infrared dye-conjugated streptavidin. As a loading control, RPS17 protein was subjected to Western blotting, and total RNA was stained with methylene blue. (D) Polysome profiling under the indicated conditions. (E) Western blotting and RNA dot blotting for proteins and RNAs along the fractions of polysome profiling (see D for the corresponding fraction numbers). Desthiobiotinylated proteins and RNAs were detected by infrared dye-conjugated streptavidin. Total RNA was stained with methylene blue. (F) Scatter plot of the ribosome footprints from each mRNA in standard Ribo-Seq. The results in the presence and absence of H 2 O 2 were compared. (G) SYPRO Ruby staining of proteins purified after streptavidin pulldown and elution under the indicated conditions. (H) Fractions of usable reads (reads after deduplication and removal of noncoding RNA-mapped reads) in standard Ribo-Seq and cytosol APEX-Ribo-Seq under the indicated conditions. (I) Distribution of ribosome footprint length under the indicated conditions. (J) Metagene plots of ribosome footprints (the 5′-end positions) around the start codon under the indicated conditions. The data for the 29-nt footprints are shown. (K) Fraction of the frame position for the 5′ end of the footprint (29 nt) under the indicated conditions. (L) Scatter plot of the ribosome footprints from each mRNA in cytosol APEX-Ribo-Seq replicates. (M) Immunofluorescence analysis of the indicated proteins (detected by V5) and desthiobiotinylated proteins/RNAs (detected with Alexa Fluor 555-conjugated streptavidin). TOM20 was detected as a mitochondrial marker. The scale bar represents 10 μm. (N) Fraction of the frame position for the 5′ end of the mitoribosome footprint (32 nt) in matrix APEX-Ribo-Seq. TPM, transcripts per million; r , Pearson’s correlation coefficient; RPM, reads per million reads. For H, K, and N, the means (bars), s.d.s (errors), and individual replicates (n = 2, points) are shown.
Article Snippet: The gels were stained with SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific, S12001) and imaged using a Pharos FX imaging system (Bio-Rad).
Techniques: Construct, Expressing, Immunofluorescence, Staining, Western Blot, Control, Purification, Marker
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