Figure 2.

Proteasome Inhibition or Expression of Mutant W58A-USP14 Enhances the Association of USP14 with HSC70

Affinity purification and bioinformatic analysis of USP14-interacting partners were performed as depicted in Figure S2.

(A) The identified USP14-interacting partners are depicted figuratively where most of the hits constitute the 26S proteasome complex and have previously been reported. The interaction of USP14 with HSPA8/HSC70 is depicted separately.

(B and C) SH-SY5Y cell lines (B) stably expressing Flag-HA-USP14 and Flag-HA-GFP, (C) transiently expressing wild-type and mutant USP14 construct (WT-USP14 and W58A-USP14) were analyzed by immunoblotting for HSC70 and Flag expression. Actin served as loading control.

SH-SY5Y cells overexpressing various USP14 constructs were treated with drugs and analyzed for the interaction with HSC70.

(D) Flag-WT-USP14 and Flag-W58A-USP14 constructs.

(E) Flag-WT-USP14 and treatment with the proteasome inhibitor, bortezomib (0.5 μM, 6 h). Treatment with DMSO was used as a negative control.

(F) Flag-WT-USP14 or Flag-W58A-USP14 co-expressed with GFP-WT-HSC70 were treated with MG132 (20 μM, 6 h) or DMSO (negative control) and subjected to IP with anti-Flag antibodies.

The immunoprecipitated complexes were analyzed by IB for HSC70 and Flag (D and E) and for GFP, and Flag (F) values represent means ± SEM, n = 3. (B) p value was calculated by student's t-test and (C) one-way ANOVA. *p ≤ 0.05; ****p ≤ 0.0001.

FASP, filter-aided sample preparation; nanoLC-MS/MS, nano-liquid chromatography coupled tandem mass spectrometry; Co-IP, co-immunoprecipitation; SAINT, significance analysis of interactome; USP14, ubiquitin-specific protease-14; WT, wild-type; IB, immunoblotting; HSPA8/HSC70, heat shock cognate 71 kDa protein; HA, hemagglutinin tag; GFP, green fluorescent protein.