β5c  (Cell Signaling Technology Inc)

Journal: Journal of Medicinal Chemistry

Article Title: α‑Aminoboronic Acid Moieties in Boro Dipeptides Modulate Proteasome Subunit Selectivity and Provide Access to Compounds with Potent Anticancer and Anti-Inflammatory Activity

doi: 10.1021/acs.jmedchem.5c02548

Figure Lengend Snippet: Effect of compounds 5b , 5c , and 5j – l , as well as subunit selective control compounds on the secretion of cytokines in LPS-stimulated PBMCs. All compounds were tested at 250 nM. The cells were pretreated for 1 h with inhibitors, followed by the addition of LPS (1 μg/mL). As the negative control (designated by ‘DMSO’ in figure legends), cells were treated only with DMSO, followed by the addition of LPS (1 μg/mL). The concentrations of cytokines were determined in the supernatants after additional 24 h treatment. The results are represented as means ± SD of four independent experiments ( N = 4). Statistical significance between untreated controls versus treated was calculated using one-way ANOVA post hoc Dunnett’s test. A p -value of less than 0.05 was considered significant (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05).

Article Snippet: Membranes were blocked with 5% BSA in 1 × TTBS for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against: β5i (1:1000; #13726), β5c (1:1000; #12919), and β1c (1:1000; #13267; all from Cell Signaling Technology), β1i (1:1000; #ab187645; all from Abcam), β2c (1:1000, #ab154745), and β2i (1:1000, #ab183506), and β-actin (1:5000; #A5316; SigmaPrestige).

Techniques: Control, Negative Control

Journal: Scientific Reports

Article Title: Cytotoxic unsaturated electrophilic compounds commonly target the ubiquitin proteasome system

doi: 10.1038/s41598-019-46168-x

Figure Lengend Snippet: Inhibition of proteasome deubiquitinases. ( a ) Examination of 20S proteasome inhibition by hit compounds. Cell lysates (25 μg) were exposed to 20 μM of each compound except for bortezomib (BZ; 50 nM) for 5 min in assay buffer followed by the addition of substrates. The substrates Suc-LLVY, Z-LLE and Ac-KQL were used to assay β5c, β1c and β2c proteasome activity; the substrates Ac-PAL and Ac-ANW were used to assay β1i and β5i immunoproteasome activity. ( b )19S proteasome preparations (10 nM) were incubated with ubiquitin rhodamine in the presence of the indicated compounds at 37 °C and fluorescence recorded; ( c ) 19S proteasome preparations (10 nM) were exposed to different compounds at 50 μM followed by labeling with HA-ubiquitin-vinylsulphone (HA-UbVS). The blots were probed with antibodies to HA, USP14 and UCHL5. Note the preferential loss of USP14 HA-UbVS labeling. The loss of recognition of USP14 following exposure to CB826 was observed in repeated experiments and remains unexplained (this phenomenon was not observed using extracts (see below)). The previously described DUB inhibitor b-AP15 was included as a reference. ( d ) Total cellular lysates from OPM-2 cells were incubated with ubiquitin rhodamine in the presence of 20 μM of the indicated compounds at 37 °C and fluorescence recorded. ( e ) OPM-2 cell extracts were incubated with compounds (20 μM) followed by HA-UbVS labeling. Filters were incubated with the indicated antibodies (also see Supplementary Fig. where compounds were used at 50 μM).

Article Snippet: We used the substrates Suc-LLVY-2R110 (β5c), Z-LLE-2R110 (β1c) and (Ac-KQL)2R110 (β2c) from AAT Bioquest (Sunnyvale, CA).

Techniques: Inhibition, Activity Assay, Incubation, Ubiquitin Proteomics, Fluorescence, Labeling