Fig 1: SENP1 regulates SUMOylation of PKC and changes phosphorylation of PKC substrate.(a) SENP1 deSUMOylates PKC in vivo. CHO-K1 cells were transfected with HA-tagged PKC, Flag-tagged SUMO1, RGS-tagged SENP1 or its catalytically inactive SENP1m as indicated. Cell lysates were precipitated with anti-HA antibody (left) or anti-Flag antibody (right; IP) and analysed by immunoblotting using anti-SUMO1, HA (IB). Whole-cell lysates were immunoblotted (IB) with anti-SUMO1, HA and RGS antibodies as indicated. (b) The SUMOylated form of PKC was determined by immunoprecipitation with anti-PKC antibodies followed by western blotting with anti-SUMO1 antibodies in SENP1+/+ (WT) and SENP1−/− (KO) MEF cells. (c) Neurons were untreated (−) or treated (+) with kainate (KA, 200 μM, 1 min) and immunoprecipitated using anti-PKC antibodies (left panel) or anti-SUMO1 antibodies (right panel), followed by immunoblotting using anti-SUMO1 or anti-PKC antibodies as indicated. (d) Kainate enhanced phosphorylation of GlyR β subunit by PKC. Cultured spinal cord neurons were untreated (−) or treated (+) with kainate (KA, 200 μM, 1 min) and cell lysates subject to immunoprecipitation using anti-GlyR β subunit antibody, followed by immunoblotting using anti-phospho-(Ser) PKC substrate (upper panel) or anti-GlyR β subunit (lower panel) antibodies, respectively, as indicated. Note: GlyR β subunit-specific bands were around 58 kDa, in part overlapping with the IgG heavy chain either in upper or lower panels. Data are representative of at least three independent experiments. (e–g) Total and phosphorylated MARCKS were determined by immunoblotting in spinal cord neurons treated with kainate (KA, 200 μM, 1 min), PMA (1 μM, 1 h) or both as indicated (e), SENP1+/+ (WT) and SENP1−/− (KO) MEF cells treated with either PMA or not as indicated (f) and tissue homogenates from E13.5 SENP1+/+ (WT) and SENP1−/− (KO) embryos (g). Glyceraldehyde 3-phosphate dehydrogenase was used as a loading control. (h) PKC activity was determined by enzyme-linked immunosorbent assay-based relative kinase assays in tissue homogenates from E13.5 SENP1+/+ (WT) and SENP1−/− (KO) embryos and was measured using the PKC kinase activity kit (Enzo) according to the manufacturer’s instructions. Data are means±s.e.m. from three experiments. *P<0.05 compared with WT by Student’s t-test P=0.0334.