anti profilin 1  (Cell Signaling Technology Inc)

Journal: Molecular Systems Biology

Article Title: Proteome-scale characterisation of motif-based interactome rewiring by disease mutations

doi: 10.1038/s44320-024-00055-4

Figure Lengend Snippet: ( A ) Left: Network of KEAP1 and its found prey proteins. Interactions and corresponding mutations disrupted by the mutation are indicated in blue, enhanced in red and neutral effects in grey. A green dot indicates previously reported interacting prey proteins and a straight line if the domain’s binding motif is found in the binding peptide. The circle size encodes the confidence level of the domain-mutation pairs. Right: Consensus motif of KEAP1 KELCH as established with the HD2 library and the aligned SQSTM1 peptide with the P348L mutation site indicated in blue. ( B ) FP-monitored displacement curves for KEAP1 KELCH domain and the SQSTM1 343-356 wild-type and P348L mutant peptides. Measurements were in technical triplets and displayed is the mean with standard deviation. ( C ) GFP trap of GFP-KEAP1 and probing for co-immunoprecipitation of wild-type/P348L Myc-SQSTM1 in HeLa cells ( n = 3). ( D ) Left: Partial network of KPNA4 and its found prey proteins. The network shows interactions with previous literature support and interactions associated with mutations having a significant effect on binding (Mann–Whitney test: p value ≤0.001). Colouring and size as in ( A ). Right: Consensus motif of KPNA4 ARM as established with the HD2 library and the aligned CDC45 and ABRAXAS1 peptides with the mutation sites indicated in blue. ( E ) Displacement curves for KPNA4 ARM domain with wild-type/R157C CDC45 152-166 and wild-type/R361Q ABRAXAS1 349-364 peptides. Measurements were in technical duplicates and displayed is the mean with standard deviation. ( F ) GFP trap of EGFP ( n = 3), wild-type EGFP-CDC45 ( n = 3) or mutant R157C EGFP-CDC45 ( n = 2) and probing for co-immunoprecipitation of T7-KPNA7 in HEK293T cells. ( G ) Displacement curves for KPNA7 ARM domain with wild-type/R157C CDC45 152-166 , sampling the major pocket. Measurements were in technical triplets and displayed is the mean with standard deviation. ( H , I ) Representative images of the localisation of wild-type and mutant EGFP-tagged (R361Q) ABRAXAS1 and (R157C) CDC45 in relation to the nuclear Hoechst staining ( n = 3 for all except ABRAXAS1 wild-type for which n = 2). The full images are provided in Appendix – . .

Article Snippet: For visualisation, mouse anti-Myc antibody (1:1000, Thermo Scientific), mouse anti-Flag antibody (1:5000, Sigma Aldrich), rabbit anti-T7 (1:1000, Cell signalling), rabbit anti-GFP antibody (1:5000 in-house) and mouse anti-GFP (1:1000, Roche) antibody were used and the membranes incubated overnight at 4 °C with the antibodies in 2.5% milk in 1xPBS with 0.1% Tween-20.

Techniques: Mutagenesis, Binding Assay, Standard Deviation, Immunoprecipitation, MANN-WHITNEY, Sampling, Staining

Journal: Molecular Systems Biology

Article Title: Proteome-scale characterisation of motif-based interactome rewiring by disease mutations

doi: 10.1038/s44320-024-00055-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: For visualisation, mouse anti-Myc antibody (1:1000, Thermo Scientific), mouse anti-Flag antibody (1:5000, Sigma Aldrich), rabbit anti-T7 (1:1000, Cell signalling), rabbit anti-GFP antibody (1:5000 in-house) and mouse anti-GFP (1:1000, Roche) antibody were used and the membranes incubated overnight at 4 °C with the antibodies in 2.5% milk in 1xPBS with 0.1% Tween-20.

Techniques: Recombinant, Plasmid Preparation, Cloning, Produced, Sequencing, Magnetic Beads, Protease Inhibitor, Staining, Marker, Gel Extraction, Picogreen Assay, Affinity Chromatography, Purification, Transfection, Cell Culture, Software, Microscopy