Fig. 6: SUMOylation dampens the K48-linked ubiquitination of ACE2.

a 293 T cells were transfected with plasmids encoding Flag-ACE2 and HA-TOLLIP, and treated with indicated concentrations of 2-D08 for 24 h. The cells cultured with Baf A1 (0.2 μM) for 6 h were immunoprecipitated with α-FLAG M2 beads and immunoblotted with anti-HA. b Domain organization of TOLLIP protein. c Co-immunoprecipitation and immunoblot analysis of 293 T cells expressing TOLLIP mutants along with HA-ACE2. d Mass spectrometry analysis of a K48-linked ubiquitin peptide of purified ACE2. e 293 T cells transfected with plasmids expressing Flag-ACE2 and HA-K48-linked ubiquitin were treated with indicated concentration of 2-D08 for 24 h. The lysates were subjected to immunoprecipitation and immunoblot analysis. f Calu-3 cells were transfected with scrambled or PIAS4-specific siRNAs. The lysates harvested after Baf A1 (0.2 μM) treatment for 6 h, were immunoprecipitated and analyzed by immunoblotting using K48-linkage specific polyubiquitin antibody and anti-ACE2 antibody. g Immunoprecipitation and immunoblot analysis of 293 T cells expressing Flag-ACE2 (WT or K187R) and HA-K48-linked ubiquitin in the presence of Myc-PIAS4. h Mass spectrometry analysis identified a ubiquitination site on ACE2 at K187. i 293 T cells expressing HA-TOLLIP and wild-type (WT) Flag-ACE2 or its K187R mutant, followed by immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA. j 293 T cells were transfected with WT Flag-ACE2 or its K187R mutant and treated with 2-D08 for indicated concentrations. The protein levels of Flag-ACE2 were analyzed by immunoblot. k Quantification of the protein levels in j. In k, all error bars, mean values ± SEM, P-values are determined by unpaired two-tailed Student’s t-test of n = 3 independent biological experiments. For a, c, e–g, i, and j, similar results are obtained by three independent biological experiments. Source data are provided as a Source data file.