mouse monoclonal mcp 1 antibody  (R&D Systems)

Journal: Cell reports

Article Title: Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10.

doi: 10.1016/j.celrep.2023.113100

Figure Lengend Snippet: Figure 1. Pirh2 and KLHDC10 directly bind to Ala-tails (A) Domain arrangement of human Pirh2: NTM, N-terminal module; RING, really interesting new gene domain; CTD, C-terminal domain. (B) In vitro GST pull-down assay using the specified GST-Pirh2 truncation constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (C) AlphaScreen assay to assess Pirh2-Ala-tail binding. Reactants were GST fusions with Pirh2 fragments containing both the NTM and RING domains (Pirh21–195) or the NTM alone (Pirh21–137) and a biotinylated Ala-tail peptide (MDELYKAAAAAA). Proximity-induced fluorescence signal (see STAR Methods) was monitored in the presence of increasing amounts of a competing, non-biotinylated Ala-tail peptide to determine IC50 values from the dose-response curve. Each data point is a technical triplicate, and error bars represent ± SD. The y axis presents the normalized AlphaScreen signal as arbitrary fluorescence units (AFUs). (D) Domain arrangement of human KLHDC10. (E) In vitro GST pull-down assay using the GST-KLHDC10 and GST-KLHDC2 constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (F) AlphaScreen assay to assess KLHDC10-Ala-tail binding. As in (C) but using GST-KLHDC10 and GST-KLHDC10b1-88–442 instead. See also Figures S1, S2, S10, and S11.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-GFP Roche Cat# 11814460001; RRID: AB_390913 Goat polyclonal anti-GST GE healthcare Cat# 27–4577; RRID: AB_771432 Mouse monoclonal anti-FLAG M2 Sigma-Aldrich Cat# F1804; RRID: AB_262044 Rabbit polyclonal anti-GRP94 Cell Signaling Technology Cat# 2104: RRID: AB_823506 Rabbit polyclonal anti-Pirh2/Rchy1 Abcam Cat# ab189247 Goat polyclonal anti-mouse IgG (H + L)-HRP Dianova Cat# 115-035-146; RRID: AB_2307392 Goat polyclonal anti-rabbit IgG (H + L)-HRP D Dianova Cat# 11-035-144; RRID: AB_2307391 Rabbit polyclonal anti-goat IgG (H + L)-HRP Dianova Cat# 305-035-045; RRID: AB_2339403 Bacterial and virus strains E. coli BL21 (DE3) E. Schiebel lab N/A E. coli DH5aplha E. Schiebel lab N/A Chemicals, peptides, and recombinant proteins Protino Glutathione Agarose 4B Macherey-Nagel Cat# 745500.10 EDTA free protease inhibitor mixture Roche Cat# 04693159001 Glutathione, reduced Sigma-Aldrich Cat# Y0000517 Lipofectamine 3000 Invitrogen Cat# L3000015 GFP-Trap magnetic agarose beads ChromoTek Cat# gtma-20 MG132 Sigma-Aldrich Cat# C2211-5MG Biotin-MDELYKAAAAAA Biosyntan N/A MDELYKAAAAAA Genscript N/A MDELYKAAAAAA-NH2 Genscript N/A MDELYKAAAAA Genscript N/A MDELYKAAAA Genscript N/A MDELYKAAA Genscript N/A MDELYKAA Genscript N/A MDELYKA Genscript N/A DELYKAAAAA Genscript N/A ELYKAAAAA Genscript N/A LYKAAAAA Genscript N/A YKAAAAA Genscript N/A KAAAAA Genscript N/A MDELYKAAAAAG Genscript N/A MDELYKAAAAAD Genscript N/A MDELYKAAAAAL Genscript N/A MDELYKLAAAAA Genscript N/A MDELYKALAAAA Genscript N/A MDELYKAALAAA Genscript N/A MDELYKAAALAA Genscript N/A (Continued on next page) 16 Cell Reports 42, 113100, September 26, 2023

Techniques: In Vitro, Pull Down Assay, Construct, Western Blot, Amplified Luminescent Proximity Homogenous Assay, Binding Assay

Journal: Cell reports

Article Title: Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10.

doi: 10.1016/j.celrep.2023.113100

Figure Lengend Snippet: Figure 5. Validation of residues implicated in Ala-tail degron recognition by Pirh2 and KLHDC10 (A) In vitro GST pull-down assay using GST-Pirh21–195 WT and mutant constructs as indicated and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti- GFP immunoblots are shown. (B) GFP or GFP-Ala6 and WT or mutant full-length FLAG-Pirh2 were expressed in HeLa cells. Interaction between GFP-Ala6 with Pirh2 and GFP-Ala6 ubiquitylation was analyzed by GFP IP followed by anti-FLAG and anti-GFP immunoblots. The dashed line indicates that the blot image has been spliced. (C) As in (A), but for GST-KLHDC10b1-88–442 WT and indicated mutant constructs. (D) As in (B), but for WT and mutant full-length KLHDC10 constructs in HeLa Pirh2-knockout cells and including anti-ubiquitin blot.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-GFP Roche Cat# 11814460001; RRID: AB_390913 Goat polyclonal anti-GST GE healthcare Cat# 27–4577; RRID: AB_771432 Mouse monoclonal anti-FLAG M2 Sigma-Aldrich Cat# F1804; RRID: AB_262044 Rabbit polyclonal anti-GRP94 Cell Signaling Technology Cat# 2104: RRID: AB_823506 Rabbit polyclonal anti-Pirh2/Rchy1 Abcam Cat# ab189247 Goat polyclonal anti-mouse IgG (H + L)-HRP Dianova Cat# 115-035-146; RRID: AB_2307392 Goat polyclonal anti-rabbit IgG (H + L)-HRP D Dianova Cat# 11-035-144; RRID: AB_2307391 Rabbit polyclonal anti-goat IgG (H + L)-HRP Dianova Cat# 305-035-045; RRID: AB_2339403 Bacterial and virus strains E. coli BL21 (DE3) E. Schiebel lab N/A E. coli DH5aplha E. Schiebel lab N/A Chemicals, peptides, and recombinant proteins Protino Glutathione Agarose 4B Macherey-Nagel Cat# 745500.10 EDTA free protease inhibitor mixture Roche Cat# 04693159001 Glutathione, reduced Sigma-Aldrich Cat# Y0000517 Lipofectamine 3000 Invitrogen Cat# L3000015 GFP-Trap magnetic agarose beads ChromoTek Cat# gtma-20 MG132 Sigma-Aldrich Cat# C2211-5MG Biotin-MDELYKAAAAAA Biosyntan N/A MDELYKAAAAAA Genscript N/A MDELYKAAAAAA-NH2 Genscript N/A MDELYKAAAAA Genscript N/A MDELYKAAAA Genscript N/A MDELYKAAA Genscript N/A MDELYKAA Genscript N/A MDELYKA Genscript N/A DELYKAAAAA Genscript N/A ELYKAAAAA Genscript N/A LYKAAAAA Genscript N/A YKAAAAA Genscript N/A KAAAAA Genscript N/A MDELYKAAAAAG Genscript N/A MDELYKAAAAAD Genscript N/A MDELYKAAAAAL Genscript N/A MDELYKLAAAAA Genscript N/A MDELYKALAAAA Genscript N/A MDELYKAALAAA Genscript N/A MDELYKAAALAA Genscript N/A (Continued on next page) 16 Cell Reports 42, 113100, September 26, 2023

Techniques: Biomarker Discovery, In Vitro, Pull Down Assay, Mutagenesis, Construct, Western Blot, Knock-Out, Ubiquitin Proteomics

Journal: Cell reports

Article Title: Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10.

doi: 10.1016/j.celrep.2023.113100

Figure Lengend Snippet: Figure 6. Conservation of residues impli- cated in Ala-tail binding among Pirh2 homo- logs (A) Multiple sequence alignment for Pirh2 homologs. The blue boxes highlight residues predicted to sta- bilize the Ala-tail peptide at the binding pocket in Pirh2 that are shown in Figure 3D and correspond- ing residues in the homologs. (B) Residues conserved in Pirh2 homologs mapped onto the AlphaFold structure of human Pirh2 (Uni- Prot: Q96PM5) using ConSurf webserver. The color key for variable to conserved residues is shown below. The Ala6 peptide is shown in stick repre- sentation at the Ala-tail-binding site. (C) AlphaFold models for Pirh2 homologs in (A) are superposed. Backbone of each protein is in ribbon representation in different colors, and human Pirh2 is represented with transparent surface. (D) In vitro GST pull-down assay using GST-tagged full-length Pirh2 homolog from S. pombe and re- combinantly purified GFP, GFP-Ala6, GFP-Thr6, or GFP-(Ala-Thr)3. Anti-GST and anti-GFP immuno- blots are indicated. See also Figures S10 and S11.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-GFP Roche Cat# 11814460001; RRID: AB_390913 Goat polyclonal anti-GST GE healthcare Cat# 27–4577; RRID: AB_771432 Mouse monoclonal anti-FLAG M2 Sigma-Aldrich Cat# F1804; RRID: AB_262044 Rabbit polyclonal anti-GRP94 Cell Signaling Technology Cat# 2104: RRID: AB_823506 Rabbit polyclonal anti-Pirh2/Rchy1 Abcam Cat# ab189247 Goat polyclonal anti-mouse IgG (H + L)-HRP Dianova Cat# 115-035-146; RRID: AB_2307392 Goat polyclonal anti-rabbit IgG (H + L)-HRP D Dianova Cat# 11-035-144; RRID: AB_2307391 Rabbit polyclonal anti-goat IgG (H + L)-HRP Dianova Cat# 305-035-045; RRID: AB_2339403 Bacterial and virus strains E. coli BL21 (DE3) E. Schiebel lab N/A E. coli DH5aplha E. Schiebel lab N/A Chemicals, peptides, and recombinant proteins Protino Glutathione Agarose 4B Macherey-Nagel Cat# 745500.10 EDTA free protease inhibitor mixture Roche Cat# 04693159001 Glutathione, reduced Sigma-Aldrich Cat# Y0000517 Lipofectamine 3000 Invitrogen Cat# L3000015 GFP-Trap magnetic agarose beads ChromoTek Cat# gtma-20 MG132 Sigma-Aldrich Cat# C2211-5MG Biotin-MDELYKAAAAAA Biosyntan N/A MDELYKAAAAAA Genscript N/A MDELYKAAAAAA-NH2 Genscript N/A MDELYKAAAAA Genscript N/A MDELYKAAAA Genscript N/A MDELYKAAA Genscript N/A MDELYKAA Genscript N/A MDELYKA Genscript N/A DELYKAAAAA Genscript N/A ELYKAAAAA Genscript N/A LYKAAAAA Genscript N/A YKAAAAA Genscript N/A KAAAAA Genscript N/A MDELYKAAAAAG Genscript N/A MDELYKAAAAAD Genscript N/A MDELYKAAAAAL Genscript N/A MDELYKLAAAAA Genscript N/A MDELYKALAAAA Genscript N/A MDELYKAALAAA Genscript N/A MDELYKAAALAA Genscript N/A (Continued on next page) 16 Cell Reports 42, 113100, September 26, 2023

Techniques: Binding Assay, Sequencing, In Vitro, Pull Down Assay, Western Blot