Fig. 1

USP3 deubiquitinates REST and extends its half-life. (A) SH-SY5Y cells were treated with the indicated concentrations of proteasomal inhibitor (MG132), lysosomal inhibitor (CQ), or inhibitor of ubiquitin activating enzyme (TAK-243) for 6 h prior to harvest. Immunoblotting was performed with the indicated antibodies. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control band (REST/GAPDH) and presented below the blots. (B) SH-SY5Y cells were transfected with increasing concentrations of Flag-USP3 and treated with MG132 for 6 h prior to harvest. A TUBEs assay was performed to assess the ubiquitination status of the REST protein in mock control and Flag-USP3 transfected cells. Cell lysates were immunoprecipitated with TUBEs antibodies, followed by immunoblotting with the indicated antibodies. (C) The ubiquitination and deubiquitination of endogenous REST were analyzed by transfecting SH-SY5Y cells with Flag-USP3, Flag-USP3CS, or sgRNA targeting USP3 followed by immunoprecipitation (IP) with an anti-REST antibody and immunoblotting with an anti-ubiquitin antibody. The cells were treated with MG132 for 6 h prior to harvest. (D) The ubiquitination and deubiquitination of ectopically expressed Myc-REST were analyzed by transfecting HEK293 cells with Flag-USP3 and Flag-USP3CS (E) Flag-USP3 and sgRNA targeting USP3(F) treatment of DUB-inhibitor PR-619 for 48 h in the HEK293 cell line prior to harvest, followed by IP with a Myc antibody and immunoblotting with an anti-ubiquitin antibody. The relative protein expression of REST-(Ub)n with respect to input REST for (C-F) was quantified using ImageJ software and represented as (REST-(Ub)n/REST) below the blot. (G-H) The knockdown effect of USP3 and reconstitution of either (G) Flag-USP3 or (H) Flag-USP3CS in USP3 depleted neuroblastoma on the half-life of endogenous REST in SH-SY5Y cells. CHX (150 µg/mL) was administered for the indicated time, and the cells were then harvested for western blotting with the indicated antibodies