Journal: International Journal of Molecular Sciences

Article Title: The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

doi: 10.3390/ijms252111650

Figure Lengend Snippet: Altered abundance of 26S proteasome subunits and associated factors in rpn10-2 1 .

Article Snippet: To examine the association of RPN13 with proteasome complexes, the latter were separated using 4% native PAGE and further separated via second-dimensional SDS-PAGE following immunoblotting using rabbit polyclonal antisera against recombinant Arabidopsis RPN13 (custom-made by Genesis Biotech, Taipei, Taiwan) or moss 20S proteasomes (a kind gift from Dr. Pirre-Alain Girod).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

doi: 10.3390/ijms252111650

Figure Lengend Snippet: Arabidopsis RPN13 interacts with RPN2a and RPN2b. ( A ) RPN13 is readily pulled down by GST-fused RPN2a or RPN2b. ( B ) Coexpression of AD-fused RPN13 with BD-fused RPN2a or RPN2b activates the HIS3 reporter, as shown by histidine auxotrophic growth. ( C ) Coexpression of AD-fused wild-type RPN13, site-specific-variant RPN13-101A, or RPN13-22A with BD-fused RPN2a or RPN2b activates the HIS3 reporter, as shown by histidine auxotrophic growth. ( B , C ) Positive (+) and negative (−) controls are p53-SV40 (SV40 T-antigen) and LAMIN (lamin C)-SV40 protein pairs, representing known interacting and non-interacting partners. ( D ) Wild-type RPN13 (Wt) and RPN13 variants A47 and Q70, but not R67 and R88, are readily pulled down by GST-fused RPN2a or RPN2b. ( A , D ) Amounts of prey and bait used in pull-down assays are 5 μg for all RPN13 variants and 35 μg, 350 μg, and 320 μg for GST, GST-RPN2a, and GST-RPN2b, respectively. One-hundredth of the input prey (50 ng) and one-tenth of the pull-down products were analyzed by immunoblotting against α-T7. One-tenth of the pull-down products (Baits) was examined by staining with Brilliant Blue R to confirm approximately equivalent immobilization. The pull-down product against GST alone was analyzed as a negative control.

Article Snippet: To examine the association of RPN13 with proteasome complexes, the latter were separated using 4% native PAGE and further separated via second-dimensional SDS-PAGE following immunoblotting using rabbit polyclonal antisera against recombinant Arabidopsis RPN13 (custom-made by Genesis Biotech, Taipei, Taiwan) or moss 20S proteasomes (a kind gift from Dr. Pirre-Alain Girod).

Techniques: Variant Assay, Western Blot, Staining, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

doi: 10.3390/ijms252111650

Figure Lengend Snippet: Arabidopsis RPN13 interacts specifically with UCH2 but not with UCH1. ( A ) GST-fused UCH2 but not UCH1 readily pulled down RPN13. ( B ) GST-fused RPN13 but not RPN13-A1A2 readily pulled down UCH2. ( C ) GST-fused RPN13 could not pull down UCH1. ( A – C ) Sample amounts used in pull-down assays were 5 μg for all preys (RPN13, UCH1, and UCH2) and ~30 μg, 105 μg, 126 μg, 130 μg, and 130 μg for GST, GST-UCH1, GST-UCH2, GST-RPN13, and GST-RPN13-A1A2, respectively. One-hundredth of the input prey (50 ng) and one-tenth of the pull-down products were analyzed by immunoblotting against α-T7. One-tenth of the pull-down products (Baits) was examined by staining with Brilliant Blue R to confirm approximately equivalent immobilization. The pull-down products against GST alone were analyzed as negative controls. ( D ) Coexpression of AD-fused RPN13 with BD-fused UCH2 but not UCH1 activated the HIS3 reporter, as shown by histidine auxotrophic growth. The positive (+) and negative (−) controls used were the same as those described in .

Article Snippet: To examine the association of RPN13 with proteasome complexes, the latter were separated using 4% native PAGE and further separated via second-dimensional SDS-PAGE following immunoblotting using rabbit polyclonal antisera against recombinant Arabidopsis RPN13 (custom-made by Genesis Biotech, Taipei, Taiwan) or moss 20S proteasomes (a kind gift from Dr. Pirre-Alain Girod).

Techniques: Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

doi: 10.3390/ijms252111650

Figure Lengend Snippet: The C-terminal 246–254 region in the Arabidopsis RPN13 DEUBAD domain is critical for UCH2 interactions. ( A ) Schematic diagram shows BD-fused UCH2 (bait) and AD-fused RPN13 variants (preys), including wild-type and site-specific, C-terminal deletion, and alanine scanning mutants for Y2H assays (see the main text for details). The 330-amino-acid UCH2 coding region is boxed in green with its unique 12-amino-acid C-terminal extension designated by a red box. Coordinates for full-length proteins, mutation sites, and alanine scanning region are indicated. ( B ) Four conserved residues, E208, L209, K275, and D279, in Arabidopsis RPN13 are not involved in UCH2 binding. Similar to AD-fused wild-type RPN13, two AD-fused dual-alanine substituted (EL-AA and KD-AA) and one AD-fused quadruple-alanine substituted (ELKD-A4) RPN13 variants could still activate the HIS3 reporter when coexpressed with BD-fused UCH2. ( C , D ) When coexpressed with BD-fused UCH2, only the AD-fused C-terminal deleted RPN13 variants CΔ1 and CΔ2, but not CΔ3–6, could activate the HIS3 reporter. ( E ) When coexpressed with BD-fused UCH2, similar to AD-fused wild-type RPN13, each of the five subregion-mutated RPN13 variants (A1–A5) could activate the HIS3 reporter. ( F ) When coexpressed with BD-fused UCH2, similar to AD-fused wild-type RPN13, each of the three serial C-terminally combined subregion-mutated AD-fused RPN13 variants (A4–5, A3–5, and A2–5) could activate the HIS3 reporter. ( G ) When coexpressed with BD-fused UCH2, unlike AD-fused wild-type RPN13, all four N-terminally combined subregion-mutated AD-fused RPN13 variants (A1–2, A1–3, A1–4, and A1–5) could not activate the HIS3 reporter. ( B – G ) The positive (+) and negative (−) controls used are the same as those described in .

Article Snippet: To examine the association of RPN13 with proteasome complexes, the latter were separated using 4% native PAGE and further separated via second-dimensional SDS-PAGE following immunoblotting using rabbit polyclonal antisera against recombinant Arabidopsis RPN13 (custom-made by Genesis Biotech, Taipei, Taiwan) or moss 20S proteasomes (a kind gift from Dr. Pirre-Alain Girod).

Techniques: Mutagenesis, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

doi: 10.3390/ijms252111650

Figure Lengend Snippet: The 12-amino-acid C-terminal extension of Arabidopsis UCH2 is critical for RPN13 interactions. ( A ) Schematic diagram shows AD-fused RPN13 (prey) and BD-fused UCH1 and UCH2 variants (baits), including wild-type (BD-UCH2 and BD-UCH1), a C-terminal-deleted UCH2 (BD-UCH2-CΔ1), alanine scanning UCH2 mutants, and three C-terminally swapped UCH1 mutants (UCH1 2C , UCH1 2C-A2 , and UCH1 2C-A3 ) for Y2H assays (see the main text for details). The UCH2 coding region and its unique 12-amino-acid C-terminal extension are illustrated in the same way as in A. The 334-amino-acid UCH1 coding region is boxed in cyan blue. Coordinates for full-length proteins, deletion/swapping sites, and alanine scanning region are indicated. ( B ) When coexpressed with AD-fused RPN13, unlike BD-fused UCH1 (UCH1) and the C-terminal-deleted BD-UCH2 fusion (UCH2-CΔ1), the BD-fused wild-type UCH2 (UCH2), a C-terminal-swapped BD-UCH1 fusion (UCH1 2C ), and a BD-fused UCH2 variant harboring all-alanine substitutions in one of three subregions of the UCH2 C-terminus (UCH2-A1) could activate the HIS3 reporter. ( C ) When coexpressed with AD-fused RPN13, similar to BD-fused UCH2, two BD-fused UCH2 variants harboring all-alanine substitutions in each of two subregions of the UCH2 C-terminus (UCH2-A2 and UCH2-A3) could activate the HIS3 reporter. Also, similar to the C-terminal-swapped BD-UCH1 fusion (UCH1 2C ), when coexpressed with the AD-fused RPN13, two C-terminal-swapped BD-UCH1 variants harboring all-alanine substitutions in each of two subregions of the swapped UCH2 C-terminus (UCH1 2C-A2 and UCH1 2C-A3 ) could activate the HIS3 reporter. ( D ) When coexpressed with AD-fused RPN13, similar to BD-fused UCH2 (UCH2), each of the four BD-fused UCH2 variants harboring all-alanine substitutions in two or all three sub-regions of the UCH2 C-terminus (A1–2, A2–3, A1/A3, and A1–3) could activate the HIS3 reporter. ( B – D ) The positive (+) and negative (−) controls used are the same as those described in .

Article Snippet: To examine the association of RPN13 with proteasome complexes, the latter were separated using 4% native PAGE and further separated via second-dimensional SDS-PAGE following immunoblotting using rabbit polyclonal antisera against recombinant Arabidopsis RPN13 (custom-made by Genesis Biotech, Taipei, Taiwan) or moss 20S proteasomes (a kind gift from Dr. Pirre-Alain Girod).

Techniques: Variant Assay