PLK2 and CK-1 are candidate kinases to modulate DSP head domain phosphorylation. (a) Graph showing endogenous total DSP protein quantification from mass spectrometry data in sgNT1, sgDPM1, and sgDPM1 SERPINB5 overexpressing cell lines. Y-axis denoted Log2 fold change of intensity values. Dots represent individual biological replicates. (b) Sequencing data showing point mutation of serine at 176 position to alanine of Dsp (HUMDP1 = hDsp1 WT, as template). (c and d) Co-immunoprecipitation assay showing DSP-GFP and DSP S176A-GFP binding to PG. Quantification shows immunoprecipitated PG normalized to the respective amount of DSP pulled down. Each dot represents individual biological replicates, Unpaired Student’s t test. (e) Kinase prediction of DSP phosphorylation site on serine at position 176 (indicated by peptide sequence in the table) by online GPS 6.0 biocuckoo tool. (f) Kinase activity analysis from phospho-proteomics datasets in sgDPM1-sgNT1 and sgDPM1 SERPINB5-sgDPM1 comparison. X-axis denotes dominant enrichment scores and Y axis denotes −log10 of dominant P values. Red indicates upregulated activities in respective kinases and blue indicates downregulated activities in respective kinases. Black dots indicate non-significantly changed kinases. The data were analyzed as described in the publication (Johnson et al., 2023). (g) Phosphate-affinity western blot showing differential phosphorylation forms of DSP-584 GFP truncated protein, upon treatment with respective kinase inhibitors. White box shows lower migrating phosphorylated forms of DSP-584 upon treatment with ON12 (PLK2 inhibitor) and D446 (CK1 inhibitor). Inset marked with red box below shows a higher exposure for clarity. Source data are available for this figure: SourceData FS4.