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<p>(Endotrophin triggers adipose tissue fibrosis and metabolic dysfunction)</p>
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<p><a href="https://www.jci.org/articles/view/63930">https://www.jci.org/articles/view/63930</a></p>
<p>(Directed evolution of APEX2 for electron microscopy and Proximity Labeling)</p>
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<p>Biotin labeling protocol -From professor Lee</p>
<p><strong>Desthiobiotin-phenol labeling in live cells</strong></p>
<p>Genes were introduced into HEK-293T or U2OS cells through transient transfection with Lipofectamine 2000 (Life Technologies). After 18–24 h (transfection), the medium was changed to 1 mL of fresh growth medium containing 250 μM desthiobiotin-phenol. This culture was incubated at 37°C under 5% CO<sub>2</sub> for 30 min. Next, 100 μL of 10 mM H<sub>2</sub>O<sub>2</sub> (diluted from 30% H<sub>2</sub>O<sub>2</sub>, H1009, Sigma Aldrich, St. Louis, MO, USA) was added to each well for a final concentration of 1 mM H<sub>2</sub>O<sub>2</sub>, and the plate gently agitated for 1 min at room temperature. The reaction was then quenched by washing three times with DPBS containing 5 mM Trolox, 10 mM sodium azide, and 10 mM sodium ascorbate; then, lysis was performed for western blot analysis or fixation was performed for imaging analysis.</p>
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<p><strong>Western blot analysis of biotin-phenol labeling. </strong></p>
<p>Cells were labeled under the same conditions described for desthiobiotin-phenol labeling above. Then cells were lysed with RIPA lysis buffer containing 1× protease cocktail, 1 mM PMSF, 10 mM sodium azide, 10 mM sodium ascorbate, and 5 mM Trolox for 10 min at 4 °C. Lysates were transferred to e-tube and was clarified by centrifugation at 15,000<em>g </em>for 10 min at 4 °C before separation on a 10% SDS-PAGE gel. For blotting analysis, gels were transferred to nitrocellulose membrane and blocked with 2% (w/v) dialyzed BSA(dBSA) in TBST (0.1% Tween-20 in Tris-buffered saline) at 4 °C overnight or at room temperature for 1hr. The blots were immersed in streptavidin-HRP in 2% dialyzed BSA in TBST (1:10,000 dilution, Thermo Scientific, cat. no. 21126) at room temperature for 30-60 min and then rinsed with TBST before development with Clarity reagent (Bio-Rad) and imaging on an ImageQuant LAS 4000 (GE healthcare).</p>
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<p><strong>Fluorescence microscope imaging. </strong></p>
<p>Biotin-labeled cells were fixed with 4% paraformaldehyde solution in DPBS at room temperature for 15 min. Cells were then washed with DPBS three times and permea­bilized with cold methanol at –20 °C for 5 min. Cells were washed again three times with DPBS and blocked for 1 h with 2% dBSA in DPBS at room temperature.</p>
<p>To detect APEX2-fusion expression, primary antibody such as anti-V5 (Invitrogen, cat. no. R960-25, 1:5000 dilution) or anti-Flag (Sigma Aldrich, cat. no. F1804, 1:3000 dilution) was incubated for 1h at room temperature. After washing four times with TBST each 5min, cells were simultaneously incubated with secondary Alexa Fluor 488-goat anti-mouse IgG (Invitrogen, cat. no. A-11001, 1:1000 dilution) and streptavidin–Alexa Fluor 568 IgG (Invitrogen, cat. no. S11226, 1:1000 dilution) for 30min room temperature. Cells were then washed four times with TBST each 5min and maintained in DPBS on ice for imaging by FV1000SPD (Olympus) of UOBC in UNIST, Korea.</p>
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