Effects of depleting or overexpressing USP9x on AGS3.

<p>(A) HEK293 cells infected by the lentivirus expressing pLVX-shRNA1, USP9x-shRNA2, or USP9x-shRNA3 were lysed and the lysates were probed with anti-AGS3 (1 µg/ml), anti-GAPDH (0.2 µg/ml), and anti-β-Actin (0.1 µg/ml) antibodies, respectively. The values in the bar graph represent the averages from 6 independent western blot analyses quantified by Li-COR Odyssey Infrared Imaging System (error bars: standard deviations). A representative western blot image was shown. (B) Characterization of AGS3 antibody in immunofluorescence. Images of endogenous AGS3 in HEK293 cells transfected with a non-targeting control siRNA, or one of three different AGS3 siRNAs (siRNA1, 2 or 3; 30 nM, 48 hrs). Cells were then stained using the anti-AGS3 antibody (1 µg/ml). (C–E) Impact of overexpression of an HA-tagged USP9x (C), or the catalytic UCH domain of USP9x (D, E), on the intensity of AGS3 staining 24 hrs after transfection. For (C) and (D), cells overexpressing the relevant proteins are indicated by arrows, arrowheads and asterisks, and the same field is shown under both 20x and 63x magnification in (D). Cells were co-stained with an anti-HA antibody (1∶1000) and the anti-AGS3 (1 µg/ml) antibodies. For (E), the average intensity of AGS3 staining in 50 transfected cells expressing a comparable level of HA-UCH or the catalytically inactive UCH(C/A) mutant was further quantified as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009725#s2" target="_blank">Materials and Methods</a>,” and the value was normalized to that of the non-transfected cells. Scale bar: 20 µm.