Figure 3.

USP10 Increases the Amount of Insoluble Ubiquitinated Proteins

(A) HeLa cells were transfected with HA-USP10, GFP-α-synuclein (GFP-α-syn), and Myc-synphilin-1 (synph) plasmids. The percentages of cells with GFP/USP10-positive aggresome are presented as the mean ± SD (n = 3); **p < 0.01.

(B) USP10-KD (USP10-1) and control (NT) HeLa cells were transfected with the non-tagged USP10 and the HA-AIMP2 plasmids. The percentages of cells with HA/USP10-positive aggresomes are presented as the mean ± SD (n = 3); *p < 0.05; **p < 0.01.

(C) Neuro-2a cells were treated with 0.5 μM MG-132 or DMSO for 12 hr. The cells were stained with the anti-α-synuclein (green) and anti-USP10 (red) antibodies and with Hoechst 33258 (blue). Arrows indicate α-synuclein-positive aggresomes induced by MG-132. Asterisks indicate dead cells. Scale bars, 10 μm.

(D) HeLa cells were transfected with the HA-USP10, the GFP-CFTR-ΔF508, GFP-α-syn/synph, or the HA-AIMP2 plasmid, and NP-40-soluble fractions (SF) and NP40-insoluble fractions (ISF) were characterized by western blot (WB) using anti-GFP, anti-HA, anti-Ub, anti-p62, anti-Lamin B, and anti-β-actin antibodies.

(E) USP10-KD (USP10-1) and control (NT) HeLa cells were transfected with the HA-USP10 and the HA-AIMP2 plasmids, and SF and ISF were characterized by WB using anti-HA, anti-Ub, anti-p62, anti-USP10, anti-Lamin B, and anti-β-actin antibodies.

(F) USP10-KD (USP10-1) HeLa cells were transfected with HA-USP10 or its mutant (USP10¹⁻²⁷⁴ or USP10¹⁻²¹⁴) plasmid together with the YFP-CL1 plasmid, and whole-cell extracts (WCE), SF, and ISF were characterized by WB using anti-GFP, anti-Ub, anti-HA, anti-Lamin B, and anti-β-actin antibodies.

(G) The percentages of cells with YFP/HDAC6-positive aggresome are presented as the mean ± SD (n = 4); **p < 0.01; NS: not significant.

See also Figure S3.