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. 2014 Jun 19;5:200. doi: 10.3389/fphys.2014.00200

Figure 5.

Figure 5

Effects of sumoylation on the transcriptional activity of TonEBP. (A) Schematics of FLAG-tagged TonEBP constructs are shown at bottom: wild type TonEBP (WT), SUMO3-fused TonEBP (SUMO-WT), K556R/K603R mutant TonEBP (MT), or SUMO3-fused MT (SUMO-MT). These constructs were transfected into HEK293 cells and immunoblotted for FLAG (top). (B) HEK293 cells were transfected with empty vector or increasing amounts of the constructs shown in A along with a TonE-driven luciferase reporter, and cultured for 4 h in isotonic (ISO) or hypertonic medium (HYP). Expression of luciferase activity is show as in Figure 3. (C) HEK293 cells were transfected with empty vector or each of the constructs shown in (A). The cells were cultured for 6 h in isotonic (ISO) or hypertonic (HYP) medium. SMIT and β-actin mRNA's were measured by RPA. The radioactivity in each SMIT band was divided by that of corresponding β-actin band to correct for RNA loading. In ISO, WT was significantly different from SUMO-WT (p < 0.01) but not from SUMO-MT. Mean + s.e.m., n = 3.