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. 2020 Nov 16;10:19908. doi: 10.1038/s41598-020-76862-0

Figure 3.

Figure 3

SMAD4 modified with O-GlcNAc at Thr59, Thr62, Thr63, and Ser69 residues. (A) O-GlcNAc on endogenous SMAD4. A549 cells were grown under thiamet-G-treated and pCMV-OGT-overexpressing conditions for 48 h. Whole cell lysates were then precipitated using sWGA. Full-length blots are presented in Supplementary Fig. S4. (B) Immunoblots showing O-GlcNAc of FLAG-SMAD4 and intensified modification by pCMV-OGT overexpression. HEK293 cells transiently expressing FLAG-SMAD4 and pCMV-OGT were prepared for immunoprecipitation with anti-FLAG. Immunoprecipitates were then probed with anti-O-GlcNAc. Full-length blots are presented in Supplementary Fig. S4. (C) A sWGA lectin affinity precipitation experiment showing O-GlcNAc on FLAG-SMAD4. Plasmids encoding FLAG-SMAD4 and pCMV-OGT were transfected into HEK293 cells for 24 h. Lectin precipitates were subjected to immunoblotting with anti-FLAG. Data are given as mean ± standard error (n = 3). The significance was *p < 0.05. P values were calculated by Student’s t test. Full-length blots are presented in Supplementary Fig. S4. (D) Immunoprecipitates showing decreased O-GlcNAc modification of the FLAG-SMAD4 QM. HEK293 cells were transfected with plasmids encoding FLAG-SMAD4 WT or five types of SMAD4 mutants for 24 h and then immunoprecipitated with anti-FLAG. Data are given as mean ± standard error (n = 3). The significance was *p < 0.05. P values were calculated by Student’s t test. Full-length blots are presented in Supplementary Fig. S4.