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. 2022 Apr 21;12:843157. doi: 10.3389/fonc.2022.843157

Figure 4.

Figure 4

Effects of Tip110 expression on IκBα phosphorylation and protein stability. (A) 293T were transfected with Tip110.His and its plasmid control, treated with 20 ng/ml of TNF-α for 0, 30 min, 1, 2 4 and 24 hr then harvested for Western blotting. pcDNA3 was used to equalize the total amount of DNA among the transfection. (B) Primary mouse embryonic fibroblasts (MEF) were transfected with 50 nM Si-Ctrl or Si-Tip110, cultured for 24 hr, treated with 20 ng/ml TNF-α for 0, 5, 15, 30, 60, or 120 min. 293T were transfected with (C) IκBα.HA or (E) IκBα.Nuc.Myc and Tip110.His or Tip110ΔNLS.His, cultured for 48 hr, treated with 20 µg/ml cycloheximide (CHX) for 0, 1, 2, 3, and 4 hr, and then harvested for Western blotting. Relative IκBα protein level was normalized to β-actin and expressed as a percentage of the untreated or as individual values (D, F). (G) 293T were transfected with Tip110 or pcDNA3, cultured for 48 hr, treated with TNF-α (10 ng/ml) for 2 or 24 hr, and harvested for total RNA isolation and qRT-PCR for IκBα mRNA level. Relative IκBα mRNA was normalized to β-actin and calculated using the untreated as a reference. *P < 0.05, **P < 0.01, ***P < 0.001.