Journal: Cell reports
Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions.
doi: 10.1016/j.celrep.2022.111902
Figure Lengend Snippet: Figure 4. The CNOT11 antenna domain recognizes the C-terminal segment of GGNBP2 (A) Structure-based sequence alignment of CNOT11-binding domain of GGNBP2. Highlighted in dark and light orange are residues with high and medium conservation across the species shown. A schematic representation shows the position of the two a-helices with respect to the amino acid sequence. (B) Co-immunoprecipitation of endogenous CNOT11 with GFP-tagged truncated GGNBP2 proteins. HEK293 cells were transfected with plasmids expressing GFP-tagged human GGNBP2 fusion proteins (numbers in parentheses indicate amino acids included, FL = Full-Length, GGNBP2 C-terminal residues were replaced by an HA tag in the GFP-GGNBP2(1-625)-HA construction). Proteins were immunoprecipitated with GFP-Trap magnetic agarose beads and the co- precipitation was analyzed by western blotting. Note that CNOT11 always appears as multiple bands in cell lysates. (C) Reconstitution of the CNOT11-GGNBP2 interaction in vitro with recombinant proteins. Chromatogram from size-exclusion chromatography and corre- sponding Coomassie-stained SDS-PAGE analysis of the peak fraction shows the presence of a complex between CNOT11C and two C-terminal segments of
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-CNOT10 Proteintech Cat# 15938-1-AP; RRID:AB_2229678 Mouse monoclonal anti-GFP (clone JL-8) Takara Cat# 632381; RRID:AB_2313808 Rabbit polyclonal anti-CNOT11 (Mauxion et al.)12 N/A Rabbit polyclonal anti-CNOT7 (Mauxion et al.)12 N/A Bacterial and virus strains Escherichia coli BL21 (DE3) STAR pRARE Stratagene N/A Escherichia coli B834 Sigma-Aldrich Cat# 69041 Chemicals, peptides, and recombinant proteins Protein Assay Dye Reagent Concentrate Bio-Rad Cat# 5000006 ChromoTek GFP-Trap Magnetic Agarose Proteintech Cat# gtma ChromoTek GFP-Trap Magnetic Particles M-270 Proteintech Cat# gtd Immobilon Western HRP Substrate MerckMillipore Cat# WBKLS0100 Luminata Crescendo Western HRP Substrate MerckMillipore Cat# WBLUR0100 IgG Sepharose 6 Fast Flow Affinity Resin Cytiva Cat# 17096901 AcTEV protease Invitrogen Cat# 12575-015 Calmodulin Affinity Resin Agilent Cat# 214303-52 cOmplete protease inhibitor cocktail, EDTA-free Roche SKU# 5056489001 H. sapiens CNOT1N (residues 1-687) + N-terminal His-SUMO-tag fusion protein cleavable with SENP2 This study N/A H. sapiens CNOT10 (residues 1-714) + N-terminal His-Thioredoxin-tag fusion proteins cleavable with 3C protease This study N/A H. sapiens CNOT11 (residues 61-510) + N-terminal His-Thioredoxin-tag fusion proteins cleavable with 3C protease This study N/A H. sapiens CNOT11C (residues 325-510) + N-terminal His-SUMO-tag fusion protein cleavable with SENP2 This study N/A H. sapiens CNOT11C-GGNBP2 (residues 638-673) + N-terminal His-SUMO-tag fusion protein cleavable with SENP2 This study N/A SENP Sigma-Aldrich Cat# SAE0067 3C Protease Sigma-Aldrich Cat# SAE0045 Critical commercial assays Effectene Transfection Reagent Qiagen Cat# 301427 Beta-Glo Assay System Promega Cat# E4720 Deposited data CNOT1N-CNOT10-CNOT11 This paper PDB: 8BFI CNOT11C This paper PDB: 8BFH partial CNOT11C-GGNBP2 (residues 638-673) This paper PDB: 8BFJ Experimental models: Cell lines Human embryonic kidney 293 (HEK293) ATCC CRL1573; RRID:CVCL_0045 (Continued on next page) 14 Cell Reports 42, 111902, January 31, 2023
Techniques: Sequencing, Binding Assay, Immunoprecipitation, Transfection, Expressing, Western Blot, In Vitro, Recombinant, Size-exclusion Chromatography, Staining, SDS Page
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