Additional file 1 of Co-relation with novel phosphorylation sites of IκBα and necroptosis in breast cancer cells

Additional file 1: Supplementary Figure 1. Schematic diagram of phosphopeptide mapping. Schematic diagram was constructed for phosphopeptide mapping. Supplementary Figure 2. MALDI MS spectrum of IκBα tryptic digestion products after phosphorylation with AURKC. Peaks corresponding to phosphopeptides and peptides are marked with peptide IDs of Table 2 and fragments of Table 1. Supplementary Figure 3. Ion chromatograms of digested phosphopeptide including Serine 32. LC-quadrupole orbitrap MS/MS spectrum of precursor ion m/z 950.4053 (charge state 3+) representing the phosphorylated peptide of sequence 24-LLDDRHDpSGLDSMKDEEYEQMVK-48. The spectrum exhibits evidence of phosphorylation at serine 32, but not serine 36. Supplementary Figure 4. Inhibition of cell proliferation and apoptosis in MDM-MB231 expressing the S32A mutant. p65 transcriptional activity in MDA-MB 231 was measured by ELISA. A) Relative to NC, the S32A mutant had the strongest inhibitory effect on transcriptional activity. Similar suppression was confirmed for S63A and S262A (*p < 0.05, **p < 0.001 vs. negative control). B, C) S32A, the positive control, had a stronger effect on cancer cell proliferation and cell death than the other mutants (*p < 0.05, **p < 0.001 vs. negative control). Supplementary Table 1. Identification of a novel phosphorylation site by LC-MS. New phosphorylation sites were identified between amino acids 54–67 and 246–264. Supplementary Table 2. Confirmation of novel phosphorylation site by MALDI-TOF MS. New phosphorylation sites were confirmed using MALDI-TOF MS.