Fig 2: Unsupervised hierarchical clustering of normalized signal:noise for total proteome peptides significantly (P < .05) differently expressed in CRISPR‐edited FBXW7‐mutated cells compared to respective FBXW7 nonmutant parental cells in replicate lysates prepared for each cell line. ARK4 replicate 2 was run twice (to fill extra 10‐plex runs; both values for replicate 2 were used in final calculations). Fold change is not shown on this figure and was calculated within replicates, comparing mutant line expression to parental. Proteins significantly differentially expressed in all three ARK1 or ARK4 FBXW7‐mutant lines compared to respective parental lines are boxed. Protein names and fold change values are provided in Figure 6 and Tables S9‐S14. Arrows indicate PADI2, which was significantly differentially expressed in all three ARK1 and ARK4 FBXW7‐mutant lines compared to respective parental controls

Fig 3: NE effect on expression of PAD2 and 4 and total modified citrullination. Following NE treatment (0–500 nM, 2 h, 37°C), total cell lysates were harvested from BMDM and protein lysates (30 μg) were separated on 4%–20% PAGE, and transferred to nitrocellulose membrane. Membranes were incubated with primary rabbit monoclonal antibody for PAD2 (a), and primary mouse monoclonal antibody for PAD4 (b). To confirm equal protein loading and protein normalization, filters were stripped and reprobed with mouse monoclonal β‐actin primary antibody and secondary HRP‐conjugated anti‐mouse IgG, and developed with SuperSignalWest Pico Chemiluminescent Substrate. Band intensities, quantified by densitometry using ImageJ software, were shown in the graphs. Graphs show the band intensities (arbitrary units) of PAD 2 (c) and PAD4 (d). Data are presented as mean ± SEM; n = 3 independent experiments with three different donor subjects *p < 0.05, versus Ctrl (vehicle control). Western blots shown are representative of N = 3 COPD donors. To measure total PAD activity, we determined total protein citrullination. Human BMDM from subjects with COPD were treated with control (C) or NE (N) (200 nM, 2 h, 37°C). Total cell lysates were harvested and protein lysates (50 μg) were separated on 4%–20% PAGE, and transferred to PVDF membrane. Membranes was stained with Ponceau S (PS) and then citrulline residues were modified and detected using AMC kit (e). Relative AMC was normalized to Ponceau S first and then normalized to control treated sample (f). Data was summarized as mean ± SEM, n = 6. AMC, anti‐modified citrulline; BMDM, blood monocyte derived macrophage; COPD, chronic obstructive pulmonary disease; NE, neutrophil elastase; NS, no significant difference.