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. 2019 Dec 19;23(1):100790. doi: 10.1016/j.isci.2019.100790

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© 2019 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

USP14 Associates with XBP1u upon Proteasome Inhibition

(A) Rat primary cortical neurons were treated for 6 h with MG132 (20 μM) or DMSO, lysed, and subjected to IP utilizing anti-USP14 antibody. Right panel depicts a histogram for the densitometry ratio XBP1u (red *)-USP14 interaction in the pull down.

(B) SH-SY5Y cells overexpressing Myc-DDK-XBP1u and treated for 6 h with MG132 (20 μM) in the presence and absence of GFP-WT-USP14 were subjected to IP with anti-Flag antibody. Treatment with DMSO served as a negative control.

(C) SH-SY5Y cells overexpressing Flag-WT-USP14 or Flag-W58A-USP14 were treated for 6 h with MG132 (20 μM) or tunicamycin (2.5 μg/mL) and immunoprecipitated with anti-Flag antibody. Treatment with DMSO served as negative control. Right panel depicts a histogram for the densitometry ratio of XBP1u normalized to Flag in the pull down.

The complexes were analyzed by immunoblotting for (A) USP14 and XBP1 (XBP1u) (B) USP14, HSC70, and XBP1 (XBP1u), (C) XBP1 (XBP1u), and Flag. XBP1u-unspliced X-box-binding protein 1. (A) n = 4, (C) n = 3. p value was calculated by student's t-test (A and C). *p ≤ 0.05; ***p ≤ 0.001.