Fig. 1.

Glycogen synthase kinase (GSK)-3β negatively regulates high NaCl-induced nuclear factor of activated T cells 5 (NFAT5) transcriptional activity. Human embryonic kidney (HEK)293 cells stably expressing a luciferase reporter of NFAT5 transcriptional activity were transfected with siRNA against GSK-3β or control siRNAs for 32 h, and then osmolality was increased to 500 mosmol/kgH₂O (NaCl added) or left at 290 mosmol/kgH₂O for 16 h. We tested siRNAs from Santa Cruz Biotechnology, Cell Signaling, and Dharmacon, finding similar results. The result using the siRNA from Santa Cruz Biotechnology is displayed here. A: siRNA decreases GSK-3β protein. B: knockdown of GSK-3β significantly increases NFAT5 transcriptional activity at both 290 and 500 mosmol/kgH₂O (NaCl added) in HEK293 cells stably expressing a luciferase reporter containing a wild-type osmotic response element (ORE). C: siRNA has no significant effect at either osmolality when the reporter contains an ORE element mutated to prevent binding by NFAT5 (*P < 0.05, compared with control siRNAs, repeated-measures ANOVA, n = 3).