Fig 1: Effects on the N-myristoylated Proteome in Living Cells Identified by Chemical Proteomics(A) Log2 fold changes of co-translationally N-myristoylated proteins. Cells were processed as in Figure 2A, with YnMyr tagging as a measure of in-cell NMT engagement. Tukey box-and-whisker plots based on n = 75 and 55 (MDA-MB-231 and HeLa, respectively) NMT substrate identifications, in triplicate. Responses of six known co-translationally N-myristoylated proteins are shown for each condition. For underlying volcano plots, see Figure S4A.(B) Log2 fold changes of proteins with an N-terminal glycine, excluding NMT substrates, in cells exposed to 1–5 for 18 h. Tukey box-and-whisker plots based on n = 289 and 229 (MDA-MB-231 and HeLa, respectively) identifications in triplicates. For underlying volcano plots, see Figure S4B.(C) Hierarchical one-minus Pearson correlation clustering of co-translationally N-myristoylated proteins quantified in MDA-MB-231 (n = 75).
Fig 2: Effects on Cellular N-myristoylation, N-myristoyltransferases, and Substrate ARL1 in Living Cells(A) Myristic acid analog YnMyr-mediated detection of NMT inhibition. Top: cells pre-incubated with or without 1–5 for 30 min, then pulsed with myristic acid analog YnMyr for 18 h and lysed. Bottom: NMT inhibition results in reduced of YnMyr tagging. YnMyr-tagged proteins are ligated by CuAAC to AzTB reporter, increasing the molecular weight (MW) (+1.3 kDa) and allowing fluorescent in-gel detection.(B) Effects on N-myristoylation revealed by YnMyr tagging in MDA-MB-231 cells. Left to right: cells exposed to DMSO (-) and 1–5, in triplicate. Top to bottom: in-gel visualization of YnMyr-tagged proteins; western blot (WB) detection of NMT1, NMT2, loading control HSP90, and NMT substrate ARL1. Higher-MW form of ARL1 (dagger) indicates NMT activity. Full gels are depicted in Figure S2A.(C) Quantification of YnMyr tagging in MDA-MB-231 and HeLa cells. Tukey box-and-whiskers plot depicts relative fluorescence intensities of n = 10 bands (asterisks in B and Figure S2A). Plots based on n = 90, 90, 90, 30, 30, 30, 90, 90, 90, 30, 30, and 30 quantifications in duplicate, each individually corrected to loading control HSP90.(D) Quantification of YnMyr tagging of ARL1 in MDA-MB-231 and HeLa cells. Tukey box-and-whiskers plot depicts relative fluorescence intensity (dagger in B and Figures S2A and S3A). Plots based on n = 9, 9, 9, 3, 3, 3, 9, 9, 9, 3, 3, and 3 quantifications in duplicate, each individually corrected to loading control HSP90.(E) Sortase A-mediated detection of NMT inhibition. Top: cells exposed to DMSO or 1–5 for 18 h, then lysed. Bottom: NMT inhibition causes accrual of non-myristoylated N-terminal glycines, amendable for sortase A-mediated addition of a biotin-tagged ALPETG-Haa peptide, increasing the molecular weight by 0.8 kDa.(F) Effects on N-myristoylation revealed by sortase A-mediated peptide addition. Left to right: cells exposed to DMSO (-) and 1–5, in triplicate. Top: WB detection of NMT substrate ARL1. Higher MW form of ARL1 (double dagger) indicates NMT inhibition. Full gels are depicted in Figure S2B. Bottom: loading control HSP90.(G) Quantification of sortase A-mediated peptide addition to ARL1 in MDA-MB-231 and HeLa cells. Tukey box-and-whisker plot depicts relative fluorescence intensity (double dagger in F and Figures S2B and S3B). Plots based on n = 9, 9, 9, 3, 3, 3, 9, 9, 9, 3, 3, and 3 quantifications in duplicate, each individually corrected to loading control HSP90.
Supplier Page from MilliporeSigma for Anti-NMT1 antibody produced in rabbit