Fig. 6
S100A14 prevents GLSKGA ubiquitination by blocking phosphorylation of residues Y308 and S314. (A and B) Huh7 cells were transfected with shS100A14 or NTC, then treated for 24 h with 25 µg/ml CHX, 20 µM proteasome inhibitor MG132, or 50 µM lysosome inhibitor chloroquine (CQ). Cell lysates were immunoblotted with antibodies against S100A14 and GLSKGA, and the protein levels of GLSKGA were quantified by ImageJ (n = 3). (C) Cells were transfected as described in panel A, treated for 8 h with 20 µM MG132. Lysates were immunoprecipitated with the anti-GLSKGA antibody. Precipitates were immunoblotted to detect ubiquitin (Ub) and GLSKGA. (D) Cells were transfected as described in panel A, and lysates were subjected to immunoprecipitation by anti-GLSKGA or anti-IgG antibody. Precipitates were fractionated on gels and silver stained. The band corresponding to GLSKGA (red arrow) was excised. (E) Western blot verified the immunoprecipitation efficiency of GLSKGA, which was immunoblotted with indicated antibodies. (F) Peptides of GLSKGA bearing post-translational modifications that were detected by mass spectrometry in lysates of shS100A14 cells, but not in NTC cells. AA, amino acid residue. (G) Molecular docking to predict a potential binding mode between S100A14 (green) and GLSKGA (pink). Key residues at the interaction interface are depicted in ball-and-stick mode. Y308 and S314 at the interaction interface are shown in heavy coloring. Hydrogen bonds are displayed as yellow dashed lines, and their distances are labeled. (H) Cells were transfected as described in panel A but also with a plasmid expressing HA-tagged wild-type (WT) or mutant forms of GLSKGA, then treated for 8 h with 20 µM MG132. Cell lysates were subjected to anti-HA immunoprecipitation, and precipitates were immunoblotted for Ub. (I) Lysates from Huh7 cells expressing HA-tagged wild-type or mutant forms of GLSKGA were immunoprecipitated using anti-HA antibody, and precipitates were analyzed using antibody against S100A14 or HA tag. ***p < 0.001; ns, not significant