Skip to main content
. 2016 Mar 18;7(16):22807–22818. doi: 10.18632/oncotarget.8182

Figure 4. The interplay between TAB3 O-GlcNAcylation and phosphorylation.

Figure 4

(A) Stably WT and S408A TAB3 transfected MDA-MB-231 cells were serum starved for 6 h, and then stimulated by IL-1β for 15 min. Aliquots of cell extract were immunoprecipitated with Flag antibody and then subjected to SDS-PAGE and immunoblotted with antibodies that recognize TAB3 phosphorylated at Thr 404. The protein was detected with the Flag antibody as a loading control. Densitometry for Flag-TAB3 Thr 404 phosphorylation was normalized against total Flag-TAB3. The data were means ± SEM. *P < 0.05, statistically significant compared with the IL-1β untreated WT TAB3 transfected group; #P < 0.05, statistically significant compared with IL-1β treated WT TAB3 transfected group. (B) Immunoprecipitation obtained from the samples previously subjected to SDS-PAGE and immunoblotted with antibodies that recognized TRAF6, TAK1 and K63 linked ubiquitin (K63Ub). (C) Stably WT and T404A TAB3 transfected MDA-MB-231 cells were serum starved for 6 h, and then stimulated by IL-1β for 15 min. Aliquots of cell extract were immunoprecipitated with Flag antibody and then subjected to SDS-PAGE and immunoblotted with CTD110.6 antibody. The protein was detected with the Flag antibody as a loading control. Densitometry for Flag-TAB3 O-GlcNAcylation was normalized against total Flag-TAB3. The data were means ± SEM. *P < 0.05, statistically significant compared with the IL-1β untreated WT TAB3 transfected group; #P < 0.05, statistically significant compared with IL-1β treated WT TAB3 transfected group. (D) Serum starved MDA-MB-231 cells were pretreated with SB203580 for 5 min, and then stimulated by IL-1β for 15 min. Cell lysate were subjected to SDS-PAGE and immunoblotted with a O-GlcNAc and phospho-specific antibodies that recognizes TAB3 (gSer408 TAB3 antibody and pThr404 TAB3 antibody), phospho-specific antibody that recognizes p38 (p-p38). The detection with total p38 and GAPDH antibodies were used as the loading controls.