Journal: Molecular & Cellular Proteomics : MCP
Article Title: Whole Proteome Profiling of N-Myristoyltransferase Activity and Inhibition Using Sortase A *
Figure Lengend Snippet: Validation and high-throughput biomarker assays by Western blotting and ELISA. A , Pull-down and Western blot analysis of SrtA-labeled proteins. MDA-MB-231, Panc-1 and HeLa cells were treated with DMSO or increasing concentration of IMP-1088 NMT inhibitor, lysed and labeled with SrtA overnight; biotinylated proteins were enriched on NeutrAvidin beads, resolved by SDS-PAGE and blotted against YES1 (60 kDa), PRKACA (40 kDa), ARL1 (20 kDa) and GAPDH as loading control (36 kDa). SN: supernatant. NMT substrate proteins show concentration-dependent increase in enrichment with increasing concentration of NMT inhibitor, whereas the low molecular weight of ARL1 also allows for direct identification of the SrtA-labeled fraction in the input sample because of the gel shift induced by ligation to Biotin-ALPET. B , Streptavidin shift analysis of SrtA-labeled proteins. SrtA-labeled samples were briefly incubated with streptavidin before SDS-PAGE and blotting against ARL1 and PRKACA. SrtA biotinylated ARL1 (B-ARL1) shows a molecular weight shift induced by the Biotin-ALPET label that gets shifted by 30 kDa on Streptavidin binding. Biotinylated PRKACA (B-PRKACA) shows no apparent shift in the absence of streptavidin, but a clear shift of 30 kDa upon addition of streptavidin. These shifts match the apparent molecular weight of streptavidin alone, as shown by Ponceau staining. C , SrtA-ELISA analysis of labeled proteins. After protein precipitation to remove excess depsipeptide, 1 μg labeled protein lysate was applied to each well of a streptavidin-coated 96-well plate and incubated for 3 h at RT. After primary (anti-ARL1) and secondary (anti-rabbit HRP) antibody incubations, turnover of QuantaBlu fluorogenic HRP substrate was monitored for 30 min. Slopes were calculated from the first 10 min (linear range) after fitting to a straight line ( Y = Slope · X + Y intercept ) by nonlinear regression (Insert). Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test.
Article Snippet: Protein-based enrichment and on-bead digestion: 8 μl NeutrAvidin agarose beads and 32 μl agarose blank beads (Pierce® Control Agarose Resin, ThermoScientific) were used for every sample.
Techniques: High Throughput Screening Assay, Biomarker Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Labeling, Multiple Displacement Amplification, Concentration Assay, SDS Page, Molecular Weight, Electrophoretic Mobility Shift Assay, Ligation, Incubation, Binding Assay, Staining