Fig. 4

Phosphoproteomic analysis of proteins with commonly regulated phosphosites in DK-MG and U-118 EMP3 KOs. A Donut chart showing the distribution of phosphorylation changes common to both DK-MG and U-118 EMP3 KO cells relative to their respective controls. Commonly phosphorylated—log₂-FC ≥ 1, FDR P-value ≤ 0.05 in both DK-MG and U-118; Commonly dephosphorylated—log₂-FC ≤ -1, FDR P-value ≤ 0.05 in both DK-MG and U-118; Differentially phosphorylated—FDR P-value ≤ 0.05 but log₂-FC in opposite directions in DK-MG and U-118. B Reactome pathways enriched based on proteins with phosphosites that are dephosphorylated in both DK-MG and U-118 EMP3 KOs. Circle sizes correspond to the number of proteins associated with each term, while the color scale indicates the significance level. C Heatmap showing IPA MRs that are activated or inhibited (|activation z-score|≥ 2, P-value ≤ 0.05) in both DK-MG and U-118 EMP3 KOs and their respective activation z-scores, colored according to predicted activity (red—active; blue—inactive). D KEA of proteins with commonly dephosphorylated sites in both DK-MG and U-118 EMP3 KOs. From each kinase-substrate database, the top 5 kinases that phosphorylate substrates in the input list were identified and listed on the y-axis. Circle sizes depict substrate number, while the color scale indicates the significance level of each kinase. E RoKAI of proteins with commonly regulated sites in DK-MG (left) and U-118 EMP3 KOs (right). Upstream kinases are listed on the y-axis and ordered according to significance level. Those with -log10(FDR-adjusted P-values) = ∞ were arbitrarily scored as 15 on the x-axis to allow plotting. Circle sizes indicate substrate number, while the color scale shows predicted kinase activity (red—active; blue—inactive). F Western blots of CDK2 in control and EMP3 KO cells. G Quantification of CDK2 band intensities (mean + S.D.) normalized to β-actin and calibrated relative to control cells (n = 3; Welch’s two-tailed t-test; *P < 0.05; **P < 0.01)