β5c  (Cell Signaling Technology Inc)

Journal: PLOS ONE

Article Title: Enhancing the immunogenicity of Wilms tumor 1 epitope in mesothelioma cells with immunoproteasome inhibitors

doi: 10.1371/journal.pone.0308330

Figure Lengend Snippet: ( A ) Structure of the 20S proteasome core and β-ring. The 20S proteasome core particle comprises four rings of seven subunits: two outer rings of α-subunits and two inner rings of β-subunits. The poly-ubiquitylated proteins are translocated into the 20S core where proteolysis occurs to produce short peptides. Aminopeptidases trim the short peptides to generate antigenic peptides of optimal length for binding to MHC-I molecules. ( B ) Three different β-subunits form a β-ring with different specificity and catalytic properties. Standard proteasome (β1c, β2c, and β5c) and immunoproteasome (β1i, β2i, and β5i) cleave substrates differently, producing different peptides (immunopeptidome). Proteasome inhibitors (MG132, epoxomicin, and bortezomib) mainly inhibit β5c and β5i, but also β1c and β2c. ONX-0914, an immunoproteasome selective inhibitor, mainly inhibits β5i but also β1i. ( C ) MESO-4 was treated with the indicated proteasome inhibitors for 3 hours, then co-cultured with reporter T cells for 20 hours, followed by a cellular immunogenicity assay. Data are represented as the mean ± SD of three independent experiments and Dunnett’s multiple comparisons test was used. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Representative data results from two independent experiments are shown.

Article Snippet: Total cell extracts were resolved on 4–12% SDS-PAGE gels and analyzed by western blotting using antibodies against WT1 (clone D8I7F, #83535), β1c (PSMB6, clone E1K9O, #13267), β2c (PSMB7, clone E1LSH, #12197), β2i (MECL-1/PSMB10, clone E6R7O, #17579), β5c (PSMB5, clone D1H6B, #12919), and β5i (LMP-7/PSMB8, clone D1K7X, #13635) (Cell Signaling Technology), β1i (Santa Cruz Biotechnology, PSMB9/LMP2, clone G-3, #373996), and GAPDH as a loading control (BioLegend, #649204).

Techniques: Binding Assay, Cell Culture, Immunopeptidomics

Journal: PLOS ONE

Article Title: Enhancing the immunogenicity of Wilms tumor 1 epitope in mesothelioma cells with immunoproteasome inhibitors

doi: 10.1371/journal.pone.0308330

Figure Lengend Snippet: ( A ) Representative western blots of protein extracts from MESO-4 treated with 20 nM ONX-0914 for 3 hours (indicated by +). Protein expressions of β1c, β2c, β5c, β1i, β2i, and β5i were analyzed by western blotting using the indicated antibodies. ( B ) Quantification of 20S proteasomal β-subunit expression was analyzed by western blotting and values were normalized to control (GAPDH). Data are represented as the mean ± SD of three independent experiments and an unpaired t-test was used. ns, not significant; **, p<0.01.

Article Snippet: Total cell extracts were resolved on 4–12% SDS-PAGE gels and analyzed by western blotting using antibodies against WT1 (clone D8I7F, #83535), β1c (PSMB6, clone E1K9O, #13267), β2c (PSMB7, clone E1LSH, #12197), β2i (MECL-1/PSMB10, clone E6R7O, #17579), β5c (PSMB5, clone D1H6B, #12919), and β5i (LMP-7/PSMB8, clone D1K7X, #13635) (Cell Signaling Technology), β1i (Santa Cruz Biotechnology, PSMB9/LMP2, clone G-3, #373996), and GAPDH as a loading control (BioLegend, #649204).

Techniques: Western Blot, Expressing, 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