Journal: bioRxiv
Article Title: Capturing Translation in Action with Protein Synthesis Profiling
doi: 10.1101/2025.11.17.688896
Figure Lengend Snippet: a, Structural model of APEX2-eEF2 bound to the ribosome. The large ribosomal subunit is shown in cyan, the small subunit in green, APEX2-eEF2 in magenta, and mRNA as a black line with the red circles indicating the alkyne modification. The blue sphere marks the ∼25 nm labeling radius of APEX2. b, Expression constructs for APEX2 and APEX2-eEF2 under the TDH3 promoter. c, Western blot detection of APEX2 and APEX2-eEF2 expression. Lanes: control (empty vector), APEX2 (27 kDa), and APEX2-eEF2 (118 kDa). Tubulin (Tub) was probed as a loading control. d, Schematic of the RNA tagging workflow. Yeast cells were incubated with alkyne-phenol (30 min), followed by H₂O₂ (5 min). After quenching, total RNA was extracted, conjugated to biotin-azide via click chemistry, and enriched with streptavidin beads. Both total and enriched RNA were used for preparing Illumina sequencing libraries. e, Agarose gel analysis of total RNA from control, APEX2, and APEX2-eEF2 cells. The presence of intact 25S and 18S rRNA bands indicates high RNA quality. M, molecular weight ladder. f, Detection of alkyne-labeled RNAs by conjugation with fluorescein-azide. Total RNA from control, APEX2, and APEX2-eEF2 cells was subjected to click chemistry and analyzed by agarose gel electrophoresis. Fluorescence was detected using a Typhoon imager. g, The same gel as in F, stained with SafeStain to verify equal RNA loading. h, Quantification of fluorescein-labeled RNA signal. The bar graph shows fluorescence intensity normalized to total RNA, averaged across two independent experiments. i, Gel-shift assay of biotin-labeled RNAs incubated with anti-biotin-AF488 antibody. RNAs from control, APEX2, and APEX2-eEF2 cells were conjugated with biotin-azide, bound by antibody, and resolved on an agarose gel. Antibody-RNA complexes are indicated by the black bar. Lanes: M, molecular weight ladder; control, RNA from control cells; APEX2, RNA from APEX2-expressing cells; APEX2-eEF2, RNA from APEX2-eEF2-expressing cells; Ab, antibody only.
Article Snippet: The blot was reprobed with mouse anti-alpha-tubulin monoclonal antibody (1:8000 dilution) (DSHB Cat# 12G10) in 5% milk for 2 hours at room temperature, followed by the HRP-conjugated anti-mouse IgG secondary antibody (1:10,000 dilution) and detected as described above.
Techniques: Modification, Labeling, Expressing, Construct, Western Blot, Control, Plasmid Preparation, Incubation, Illumina Sequencing, Agarose Gel Electrophoresis, Molecular Weight, Conjugation Assay, Fluorescence, Staining, Gel Shift
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