Fig. 1

Schematic view of the microplate-based Matrix-IP method and hnRNP K peptide coverage, differential PTMs and number of proteins in the in Hepa1.6 cell interactome culture. (a) 96-well polypropylene microplates are irradiated with UV-C light for 48 h and coated with protein A/G, sealed, and kept at 4 °C prior to use. Detailed protocol is described the Methods section. (b) hnRNP K peptide coverage and localization of PTMs. Grey blocks indicate sequence covered with peptides found in MS/MS runs. Visualization of the coverage and detected modification sites were performed using CLC Sequence Viewer (CLC Bio). (c) K protein serine p284 phosphorylation (pSer284) changes in high glucose and oxidative stress conditions. Cells were seeded in 10 % FBS DMEM containing 4.5 g/L glucose. Next day medium was switched for 0.5 % FBS either with 4.5 g/L glucose or without glucose, and 24 h later cells without glucose were subjected to a challenge with 3.5 m M H₂O₂/0.1 mM Na₃VO₄ for 30 min. Cells were collected for protein extraction followed by hnRNP K IP reaction on Matrix-IP, SDS-PAGE and MS quantitative analyses. Bars represent mean values from five biological replicates and whiskers are for standard error of mean values. P-values < 0.05 were considered significant (*), (**) p-value < 0.0001. (d) Venn diagram presenting number of K protein interacting proteins in culture without, with glucose and under oxidative stress conditions. Cells were prepared as in C and IP reaction was performed with either unspecific IgG or anti-hnRNP K antibody followed by MS shotgun protein identification. K protein interactome, 178 proteins listed in Additional file 7: Table S6, was generated by subtracting IgG associated proteins from K protein specific bound proteins