Fig. 3.

SUMO enhances the nuclear localization of Notch1, interacts with Notch1 in the nucleus, and increases the levels of Notch1. a Sumoylation of Notch1 increases the nuclear localization of Notch1. COS7 cells were transfected with activated Notch1 (ΔEN) and/or His-SUMO1 (S1), or a control vector. Twenty-four hours after transfection the cells were left untreated or heat-shocked for 1 h at 42 °C followed by a 1-h recovery at 37 °C (HS). Images were taken with a Zeiss LSM510 META confocal microscope. n = 3. b Heat shock combined with transfected SUMO increases the nuclear localization of Notch1 most efficiently. Values are statistically significant at **p < 0.01 and represent averages from three independent experiments. Hundred cells in average were counted for each sample. c Notch1 is sumoylated in the nucleus. COS7 cells were heat-shocked and recovered as in (a). Nuclear fractions (NE) and cytoplasmic fractions (Cyt) were extracted and the samples were immunoblotted. GFP-Flag-NICD1 (NICD1) shows strong sumoylation in the nuclear fractions, whereas the cytoplasmic fractions show no conjugation of His-SUMO1 (S1) to NICD1. n = 3. d Consistent with (c), NICD1 cleaved from overexpressed ΔEN does not interact with His-SUMO1 (S1) in the cytoplasm, but only in the nucleus. The samples were treated as in (c), but immunoblotted with α-Notch1 C20. e COS7 cells were transfected as indicated, and  left untreated or treated with the protein translation inhibitor cycloheximide (CHX). The cells were heat-shocked at 42 °C followed by a 1-h recovery at 37 °C (HS + rec.), or  left untreated, and the samples were lysed and immunoblotted. f The levels of Notch1 are increased with increasing amounts of transfected SUMO1. COS7 cells were transfected with GFP-Flag-NICD (NICD) and different amounts of His-SUMO1 (S1), heat-shocked and recovered, lysed, and immunoprecipitated against Notch1 with protein G-sepharose beads and α-Notch1 C20, and analyzed with immunoblotting. The sumoylated form of Notch1 is indicated with an arrow or a bracket and the unmodified form with an asterisk. The lysates showed less difference in the levels of NICD, presumably due to decreased sumoylation of Notch1 in the presence of desumoylating isopeptidases. n = 3