proteasome 20s α4 subunit monoclonal antibody  (Enzo Biochem)

Journal: bioRxiv

Article Title: PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

doi: 10.1101/160739

Figure Lengend Snippet: (A) Graphic representation of the result of endogenous PA28γ SILAC IP in U2OS cells, visualized by plotting log 2 (H/L) versus log 2 (H intensity) values for all 68 human protein groups quantified by MaxQuant with a minimum of 2 unique peptides identified. Proteins specifically interacting with the bait, i.e., PA28γ, are expected to show Heavy/Light (H/L) ratios higher than 1. In contrast, proteins non-specifically binding to the beads (experimental contaminants) are expected to show H/L ratios close to 1. Proteins with ratios lower than 1 are mostly environmental contaminants, such as keratins. (B) Graphic representation of the result of the SILAC GFP-TRAP IP of GFP-FAM192A, visualized by plotting log 2 (H/L) versus log 2 (H intensity) values for all 364 protein groups quantified by MaxQuant with a minimum of 2 unique peptides identified. In both graphs, PA28γ is indicated by a red dot, FAM192A is indicated by an orange square and GFP by a green dot. The PSMA1 subunit of the 20S core proteasome is indicated by a violet diamond. Ribosomal proteins, which can be considered here as non-specific interaction partners, are indicated by grey diamonds. (C) Recombinant PA28γ and FAM192A directly interact with each other, as analyzed by native electrophoresis. After electrophoresis, the proteins were transferred on membrane and immunoprobed with specific antibodies visualized with an Odyssey infrared imaging system (LI-COR Biosciences). The arrowhead on the right indicates the formation of a complex (yellow) between purified PA28γ (green) and PIP30 (red). (D) Effect of FAM192A and/or PA28γ knock-down on the incorporation of 19S, PA28γ, and FAM192A into proteasome complexes. Proteasome complexes were immuno-purified using the MCP21 antibody (targeting the α2 subunit of 20S proteasome) from total extracts of siRNA-treated HeLa cells. Proteins were identified and quantified by nano-LC-MS/MS analysis. SD were calculated from triplicates. *: p = 0.02 (E) Quantification of PA28γ-bound 20S proteasome in wild-type and FAM192A -/- U2OS cells, as determined by co-immunoprecipitation of α4 subunit from 150 µg of total cell extract with anti-PA28γ antibodies. The right panel is constructed from three independent experiments (error bars: SD).

Article Snippet: Unless otherwise specified, the sources of the antibodies used in this study were as follows: anti-PA28γ (rabbit polyclonal, BML-PW8190, ENZO Life Sciences; rabbit polyclonal, 383900, Zymed/Life Technologies; rabbit polyclonal, PD003, MBL; mouse monoclonal, 611180, BD Transduction), anti-α2 subunit of the proteasome 20S (mouse monoclonal MCP21), anti-α4 subunit of the proteasome 20S (mouse monoclonal PW8120, ENZO Life Science) anti-GFP (rabbit polyclonal, TP401, Torrey Pines Biolabs), GFP-TRAP® beads (Chromotek), anti-coilin (mouse monoclonal 5P10 ( )) for indirect immunofluorescence, rabbit polyclonal PLA 0290, Sigma-Aldrich, for PLA and IPs, rabbit polyclonal R288 ( ) for WB), anti-β-actin (rabbit monoclonal, 13E5, Cell Signalling), anti-α-tubulin (mouse monoclonal, T9026, Sigma-Aldrich), anti-WRAP53 (A301-442A, Bethyl).

Techniques: Binding Assay, Recombinant, Electrophoresis, Imaging, Purification, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Construct

Journal: bioRxiv

Article Title: PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

doi: 10.1101/160739

Figure Lengend Snippet: (A) Inhibition by PIP30 of PA28γ-activated 20S proteasome is peptide-substrate specific. Proteasome activity was assayed in the presence of the indicated peptides and proteins and normalized by setting to 100% the value obtained in the presence of 20S proteasome and PA28γ. The assays were performed for 20 min at 37°C in a 50μl reaction mixture containing the indicated combinations of 20S proteasome (0.5µg), PA28γ (1µg), and PIP30 (4µg). The respective final concentrations of the 20S proteasome and PA28γ are set up such as the peptidase activities are not fully activated (i.e. PA28γ is limiting in the assay). At the final concentration used for PIP30, the inhibitory effect is maximal (Fig. S5A). Error bars represent deviation from the mean of technical duplicates. The figure is representative of >3 distinct experiments. Peptides used are substrates of the following peptidase activities of the 20S proteasome: Suc-LLVY-amc, Z-GGL-amc : chymotrypsin-like activity; Ac-nLPnLD-amc, Z-LLE-amc : caspase-like activity; Boc-LRR-amc, Boc-LSTR-amc : trypsin-like activity. (B) The activity of the chymotrypsin-like and trypsin-like catalytic sites of the 20S proteasome is not modified by PIP30: 20S proteasome (2 μg) was mixed in reaction buffer (Tris-HCl 20mM pH 7.5, DTT 1mM, glycerol 10%), in a final volume of 18µL, with either no protein or with PA28γ (6μg) preincubated for 5 min at RT with or without PIP30 (8.3µg). After incubation 5 min at RT, 2µL of the probe Bodipy TMR-Ahx 3 L 3 VS (1µM in reaction buffer) were added to each sample and the mixtures were further incubated 15min at 37°C. After denaturation with Laemmli buffer and electrophoresis, the labeled proteasome subunits were visualized (left panel) using a Typhoon FLA 7000 (GE Healthcare). The labeling of subunits β2 and β5 was quantified and plotted after normalization by the value of their labeling in 20S proteasome alone (right panel). In the experimental conditions used, the labeling of both subunits is roughly proportional to their activity. The figure is representative of 3 distinct experiments. (C) Comparison of the effects of PIP30 and its CK2-phosphorylated form (PIP30-Ph) on the peptidase activities of the PA28γ / 20S proteasome complex. The experiments were performed as in (A) except that only 2 μg of PIP30 and PIP30-Ph were used per assay. Error bars represent deviation from the mean of technical duplicates. The figure is representative of 3 distinct experiments.

Article Snippet: Unless otherwise specified, the sources of the antibodies used in this study were as follows: anti-PA28γ (rabbit polyclonal, BML-PW8190, ENZO Life Sciences; rabbit polyclonal, 383900, Zymed/Life Technologies; rabbit polyclonal, PD003, MBL; mouse monoclonal, 611180, BD Transduction), anti-α2 subunit of the proteasome 20S (mouse monoclonal MCP21), anti-α4 subunit of the proteasome 20S (mouse monoclonal PW8120, ENZO Life Science) anti-GFP (rabbit polyclonal, TP401, Torrey Pines Biolabs), GFP-TRAP® beads (Chromotek), anti-coilin (mouse monoclonal 5P10 ( )) for indirect immunofluorescence, rabbit polyclonal PLA 0290, Sigma-Aldrich, for PLA and IPs, rabbit polyclonal R288 ( ) for WB), anti-β-actin (rabbit monoclonal, 13E5, Cell Signalling), anti-α-tubulin (mouse monoclonal, T9026, Sigma-Aldrich), anti-WRAP53 (A301-442A, Bethyl).

Techniques: Inhibition, Activity Assay, Concentration Assay, Modification, Incubation, Electrophoresis, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes *

doi: 10.1074/jbc.M116.717652

Figure Lengend Snippet: Proteasomal activators interact with 20S/26S proteasomes forming alternative proteasome complexes. A, gel filtration assay of PA28γ proteasome complexes in BZ-treated HeLa cells compared with control. Western blotting analysis of RPT6, β2, and PA28γ in the top panel shows representative images of the protein distribution in the different fractions separated by gel filtration. The bottom panel shows the quantification of the signal for PA28γ in BZ-treated cells and controls. For clarity, the intensity of the signal has been normalized to the maximum value of each curve, and the total areas under each curve were made equal. B, analysis of interaction of proteasomal activators with the 20S proteasome using co-immunoprecipitation (IP). Lysates of phLF were treated with BZ for 6 h or with solvent, lysed, and subjected to immunoprecipitation using an antibody against the 20S subunit α4. Co-immunoprecipitated proteins as well as total protein lysate (input, 10% of total volume) were separated by SDS-PAGE. Direct interaction of proteasomal activators PA28γ and PA200 with the 20S proteasome subunit α4 was visualized via immunoblotting. Representative results of experiments in cells from three different donors are shown.

Article Snippet: 3 μl of antibody specific for 20S subunit α4 (BML-PW8120, Enzo Life Sciences) were incubated with Protein G magnetic beads (Dynabeads, Thermo Fisher Scientific) for 15 min at 1200 rpm.

Techniques: Filtration, Western Blot, Immunoprecipitation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes *

doi: 10.1074/jbc.M116.717652

Figure Lengend Snippet: Proteasomal activators interact with 20S/26S proteasomes forming alternative proteasome complexes. A, gel filtration assay of PA28γ proteasome complexes in BZ-treated HeLa cells compared with control. Western blotting analysis of RPT6, β2, and PA28γ in the top panel shows representative images of the protein distribution in the different fractions separated by gel filtration. The bottom panel shows the quantification of the signal for PA28γ in BZ-treated cells and controls. For clarity, the intensity of the signal has been normalized to the maximum value of each curve, and the total areas under each curve were made equal. B, analysis of interaction of proteasomal activators with the 20S proteasome using co-immunoprecipitation (IP). Lysates of phLF were treated with BZ for 6 h or with solvent, lysed, and subjected to immunoprecipitation using an antibody against the 20S subunit α4. Co-immunoprecipitated proteins as well as total protein lysate (input, 10% of total volume) were separated by SDS-PAGE. Direct interaction of proteasomal activators PA28γ and PA200 with the 20S proteasome subunit α4 was visualized via immunoblotting. Representative results of experiments in cells from three different donors are shown.

Article Snippet: Membranes were incubated overnight at 4 °C with one of the following antibodies against PA28γ (sc-136025, 1:1000, Santa Cruz Biotechnology; BML-PW8190-0100, 1:2000, Enzo Life Sciences), PA200 (NBP2-22236, 1:3000, Novus Biologicals), PA28α (ab155091, 1:1000, Abcam), 20S subunit α4 (BML-PW8120, 1:2000, Enzo Life Sciences), 20S subunit β5 (ab90867, 1:1000, Abcam), proteasome 20S α1–7 (ab22674, 1:1000, Abcam), Lys 48 -specific ubiquitin (05-1307, 1:1000, Merck Millipore), 19S subunit RPT5 (A303–538A, 1:5000, Bethyl Laboratories), 19S proteasome subunit RPN6 (NBP1-46191, 1:2000, Novus Biologicals), cyclin D1 (2978, 1:500, Cell Signaling) and p21 (MAB88058, 1:3000, Merck Millipore). β-Actin HRP (A3854, 1:80,000, Sigma) was used to monitor equal protein loading and for subsequent normalization of densitometric signals.

Techniques: Filtration, Western Blot, Immunoprecipitation, SDS Page