Fig. 3: Autocitrullination of PAD4, incorporation of citrulline in PAD4 and implications thereof.
From: Site-specific incorporation of citrulline into proteins in mammalian cells
a Chemical structure of Rh-PG and the fluorescence labeling of autocitrullinated PAD4. The bands at 0 min in the presence of calcium and at all the time points in the absence of calcium correspond to the basal levels of autocitrullination during the expression and purification of PAD4 from E. coli. n = 2 independent experiments, data are presented as mean value ± SD. b Expression of R372Cit PAD4 in the presence of engineered release factor, eRF1-E55D and SM60 in HEK293T cells, indicating the essential role of eRF1-E55D for efficient TAG suppression. c Coomassie stains for WT, R372Cit and R374Cit PAD4, indicating their purity. d Michaelis–Menten kinetics for WT, R372Cit, and R374Cit PAD4. n = 2 independent experiments, data are presented as mean value ± SD. e Western blot analysis of histone H3 citrullination in live HEK293T cells by WT and R374Cit PAD4. The normalization procedure is given in the “Methods” section. n = 3 independent experiments, data are presented as mean value ± SD. The samples derive from the same experiment and the blots were processed in parallel (see Supplementary Fig. 20c for a detailed explanation). f Thermal shift profiles of WT and R374Cit PAD4. Western blot images are given in Supplementary Fig. 17. The table indicates the melting temperatures (Tm). n = 2 independent experiments, data are presented as mean value ± SD. The full gels corresponding to panels (a), (b), and (e) are given in Supplementary Fig. 20. Source data for panels (a) and (d–f) are provided as a Source Data file.
