USP25 interacts with ERAD components.

<p>A) Schematics depict known domains of common (USP25(WT)) and muscle-specific (USP25(m)) isoforms of USP25 that are expressed in mammals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Denuc1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Meulmeester1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Valero1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Valero2" target="_blank">[42]</a>. B) HEK-293 cells were transfected with HA-USP25. 48 hours later cells were fixed, probed as indicated and imaged with laser confocal microscopy. Panels IA-IC are single optical plane images (1 µM) of a cell immunolabeled for ER (KDEL, endogenous marker), HA-USP25 and nucleus (DAPI). Panel IC is the merged view of panels IA (green channel), IB (red channel) and DAPI (blue channel; not shown as a separate channel). Panels II and III are merged views of other cells stained similarly to panel I. Scale bars: 10 µM. C–G) HEK-293 cells were transfected as shown. Indicated constructs were immunopurified with bead-bound antibodies. Similar results were obtained from COS-7 cells for panels B–E (not shown). All USP25 constructs used in this figure were the common isoform (USP25(WT)).</p

USP25 and HRD1 have opposing effects on CD3

<p>δ <b>protein levels and ubiquitination.</b> A) HEK-293 cells were transfected as indicated and harvested 48 hours later. Western blots are from whole cell lysates. HRD1(WT): normal HRD1; HRD1(CA): catalytically inactive HRD1, in which the catalytic cysteine is substituted by an alanine residue <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Kikkert1" target="_blank">[7]</a>. Histograms on the right: semi-quantification of data from the left and other independent experiments. Shown are means +/− standard deviations. CD3δ levels were normalized to loading control. P values from Student T tests are shown below histograms. B and C) HEK-293 cells were transfected with the indicated constructs. 48 hours post transfection, cells were treated for 6 hours with MG132 (15 µM) and HA-CD3δ was immunopurified using bead-bound anti-HA antibody after a stringent denature/renature step (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#s4" target="_blank">Materials and Methods</a> for details). Histograms: semi-quantification of bracketed ubiquitin smears from the experiment on the left and other similar, independent experiments. Shown are means +/− standard deviations. P values for panel C are from Student T-tests. </p

USP25 inhibits degradation of the ERAD substrate CD3δ

<p>. A) Western blots of whole cell lysates. Top: HEK-293 cells were transfected as indicated and treated with the proteasome inhibitor MG132 where noted (15 µM, 6 hours) before harvesting. Bottom: semi-quantification of bands from western blots shown above and other similar, independent experiments. CD3δ protein levels were normalized to loading control. Shown are means +/− standard deviations. USP25(WT): common isoform of USP25; USP25(m): muscle-specific isoform of USP25. P values from Student T-tests are shown below histograms. B) Top: HEK-293 cells were transfected with the indicated constructs and harvested 48 hours later. Shown are western blots of whole cell lysates probed with the indicated antibodies. WT: wild type USP25, C178S: the catalytic cysteine of USP25 was replaced by a serine residue <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Denuc1" target="_blank">[18]</a>, ΔUBA: UBA deleted, ΔUIM: both UIMs deleted. Bottom: semi-quantification of data from the top and two other independent experiments. CD3δ protein levels were normalized to loading control. Shown are means +/− standard deviations. P values from Student T-tests are shown below histograms. C) Top: HEK-293 cells were transfected as indicated. 48 hours post-transfection cells were treated for the indicated periods of time with 75 µg/ml cycloheximide to inhibit synthesis of new protein. Bottom: semi-quantification of western blots from the top and three other, independent experiments. CD3δ levels were normalized to loading control. Shown are means +/− standard deviations. P values are from Student T-tests of USP25 compared to vector control. D and E) HEK-293 cells were transfected with the indicated constructs. 48 hours later tagged constructs were immunopurified with bead-bound antibodies and probed as indicated.</p

USP25 regulates protein levels of the ERAD substrates APP and CFTRΔF508.

<p>A) Left: whole cell lysates of HEK-293 cells transfected with the indicated constructs. USP25 (WT) and USP25(m) are both catalytically active isoforms. Where noted, cells were treated with the proteasome inhibitor MG132 (15 µM, 6 hrs) before harvesting. Right: histograms show semi-quantification of APP signal from the left portion and other similar, independent experiments. Bracket: APP bands were quantified separately, added and normalized to loading control. Shown are means +/− standard deviations. P values from Student T-tests are shown above histograms. No statistically significant differences were observed when cells were treated with MG132. B) Left: whole cells lysates of HEK-293 cells transfected as indicated and treated 48 hours later with cycloheximide to inhibit translation of new protein. Right: semi-quantification of western blots from the right and two other independent experiments. Shown are means +/− standard deviations. APP levels were normalized to loading control. P values are from Student T-tests where APP levels in the presence of USP25(WT) were compared to APP levels in presence of vector control. C) Left: HEK-293 cells were transfected with shRNA constructs targeting different portions of endogenous USP25 (RNAi-1, 2) or scramble RNA (RNAscr-1, 2). Cells were harvested 48 hours post-transfection and probed as indicated in western blots. Trials with 72 hour-long transfections yielded similar results (not shown). Right: semi-quantification of signal from the left and other similar, independent experiments. Bracket: APP bands were quantified separately, added together and normalized to loading control. Asterisks: P<0.01 according to Student T-tests comparing RNAi-1 and RNAi-2 lanes to RNAi-scr lanes. D) HEK-293 cells were transfected with the indicated constructs and Myc-USP25 was co-immunoprecipitated 48 hours later. E and F) HEK-293 cells were transfected with the indicated constructs. Western blots of whole cell lysates. For panels D, E and F: similar results were obtained from COS-7 cells (not shown).</p