Fig. 5: ROCK2 is SUMOylated on K1007 to be activated.
From: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways
a, b Western analyses in 293T cells transfected with Myc-SUMO1 for 24 h or the indicated times. c Co-immunoprecipitation experiments using a control IgG or a ROCK2 antibody in 16HBE cells and mouse primary bronchial epithelial cells (MPBEs). d Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with Flag-ROCK2 in combination with Myc-SUMO1 in the presence of scramble or UBC9 shRNA. e In vitro SUMOylation assays in a reaction mixture containing ROCK2 recombinant protein, E1, E2, and SUMO1 and incubated at 37 °C or 4 °C for 60 min, followed by western analyses. f Western analyses in 293T cells 24 h after transfection with vector, wild-type (WT) ROCK2 or ROCK2 variants (K → R). g Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with Myc-SUMO1 and Flag-ROCK2/Flag-ROCK2(K1007R). h Western analyses in 293T cells at 24 h post-transfection with or without Myc-SUMO1, UBC9 shRNA, and Flag-ROCK2 /Flag-ROCK2(K1007R). i In vitro SUMOylation assays in a reaction mixture containing ROCK2(WT) or ROCK2(K1007R) recombinant protein, E1, E2, and SUMO1, followed by western analyses. j, k Western analyses in 293 T cells transfected with Flag-ROCK2(WT or K1007R) in the presence of IL-13 stimulation for 6 h or of Myc-caRhoA. l Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with HA-RhoA and Flag-ROCK2(WT or K1007R) after IL-13 treatment for 6 h. m 16HBE cells transfected with Flag-ROCK2(WT or K1007R), were subjected to GST pull-down assays with Rhotekin-RBD-coated beads, followed by western analyses. Experiments were repeated independently at least three times with similar results. Source data are provided as a Source Data file.
