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. 2019 Dec 1;9(12):2749–2759.

Figure 1.

Figure 1

USP37 is a direct positive regulator of SNAI1. A. Twenty-three of 68 DUBs physically associated with SNAI1. Each SFB-tagged DUB was co-transfected with MYC-SNAI1 into HEK293T cells, followed by pulldown with S-protein beads and immunoblotting with antibodies against FLAG and MYC. B. Upper panel: each SFB-tagged DUB was transfected into HEK293T cells, followed by immunoblotting with antibodies against SNAI1, FLAG, and β-actin. Lower panel: quantification of SNAI1 protein levels (normalized to β-actin). C. SFB-tagged DUBs and MYC-SNAI1 were co-transfected into HEK293T cells, followed by pulldown with S-protein beads and immunoblotting with antibodies against FLAG and MYC. D. SFB-tagged DUBs and MYC-SNAI1 or MYC-GFP were co-transfected into HEK293T cells, followed by immunoprecipitation (IP) with anti-MYC beads and immunoblotting with antibodies against FLAG and MYC. E. Left panel: HEK293T cells were co-transfected with HA-ubiquitin, MYC-SNAI1, and SFB-tagged DUBs, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. Cells were treated with 10 μM MG132 for 6 hours before collection. Before immunoprecipitation, lysates were heated at 95°C for 5 minutes in the presence of 1% SDS (for denaturing), followed by 10-fold dilution with lysis buffer and sonication. Right panel: immunoblotting of HA-ubiquitin, MYC-SNAI1, FLAG-DUBs, and HSP90 in the input. F. Upper panel: HEK293T cells were co-transfected with SNAI1, MYC-GFP, and SFB-tagged GFP or DUBs, treated with 100 μg ml-1 cycloheximide (CHX), harvested at different time points, and then immunoblotted with antibodies against SNAI1, MYC, and FLAG. MYC-GFP served as the control for transfection. LE: long exposure. SE: short exposure. Lower panel: quantification of SNAI1 protein levels (normalized to MYC-GFP). G. Upper panel: SFB-GFP, SFB-USP29, and SFB-USP37 were purified from HEK293T cells transfected with SFB-tagged GFP or DUBs, via pulldown with Streptavidin beads, followed by elution with biotin. Purified SFB-GFP, SFB-USP29, or SFB-USP37 was incubated with purified His-SNAI1, followed by pulldown with S-protein beads and immunoblotting with antibodies against SNAI1 and FLAG. Lower panel: purified proteins were analyzed by SDS-PAGE and Coomassie blue staining.