UBE4B and LSD1 double-knockdown accelerates SOD1^G85R protein degradation.

Abstract

<p>(<b>A</b>) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSD1 or UBE4B single-knockdown reduces the SOD1^G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. (<b>B</b>) Quantification of SOD1^G85R protein levels by western blotting (A). <i>n</i> = 3 (supernatant); <i>n</i> = 8 (pellet). (<b>C</b>) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. (<b>D</b>) Quantification of SOD1^G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1^G85R at each chase time point. <i>n</i> = 5; Overall <i>p</i> = 0.02 (paired <i>t</i> test, CTRL versus UBE4B and LSD1 double knockdown). Individual <i>p</i> = 0.03 (3 h), <i>p</i> = 0.003 (6 h), <i>p</i> = 0.06 (9 h), and <i>p</i> = 0.004 (12–21 h). (<b>E</b>) The half-life of SOD1^G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p