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. 2014 Apr 23;2(4):e12000. doi: 10.14814/phy2.12000

Figure 3.

Figure 3.

Effect of knock down of 14‐3‐3‐β and/or ‐ε on protein abundance of NFAT5. (A) Knock down of 14‐3‐3‐β and/or ‐ε reduces NFAT5 protein abundance. siRNAs against 14‐3‐3‐β and/or ‐ε were transfected for 3 days into HEK293 cells incubated at 300 mosmol/kg, then the medium was changed, keeping the osmolality at 300 or increasing it to 500 mosmol/kg for 8 h by adding NaCl. Upper panel is a representative Western blot. The siRNAs singly and in combination reduce NFAT5 protein abundance (mean ± SEM, n =3, *P <0.05). (B) Time course of the effect of 14‐3‐3‐β and ‐ε in combination. As in (A) except that NFAT5 protein was measured just before NaCl was increased (zero time) and after that at the intervals that are shown. Upper panel is a representative Western blot (Mean ± SEM, *P <0.05, n =3). (C) Lack of effect of 14‐3‐3‐β and/or ‐ε separately or combined on NFAT5 mRNA abundance. As in (A) except that NFAT5 mRNA was measured 16 h after NaCl was increased (P >0.05, n =3). (D–F) siRNA‐mediated knockdown of 14‐3‐3‐β and ‐ε in combination increases degradation of NFAT5 protein. siRNAs against 14‐3‐3‐β and ‐ε were transfected for 3 days into HEK293 cells incubated at 300 mosmol/kg. (D) One hundred μg/mL cycloheximide (CHX) or vehicle (DMSO control) was added for 1 h before changing to otherwise identical media kept at 300 mosmol/kg or increased to 500 mosmol/kg by adding NaCl. Media contained CHX or DMSO. NFAT5 protein was measured by Western blot at the start and end of the 8 h increase in NaCl, and the difference (Δ) was calculated. (*P <0.05, n =3). (E and F) Measurement of the rate of NFAT5 degradation by cycloheximide chase. HEK293 cells were preincubated for 7 h at 300 or 500 mosmol/kg, then 100 μg/mL cycloheximide was added for 1 h. Cells maintained in cycloheximide were harvested at the intervals shown. (*P <0.05, n =3–4). (G) Lack of coimmunoprecipitation of 14‐3‐3‐β and ‐ε with NFAT5. NFAT5‐V5 was immunoprecipitated from HEK293 cells, then the immunoprecipitates were immunoblotted for the proteins shown. CDK5 and HSP90 coimmunoprecipitate with NFAT5‐V5, but 14‐3‐3‐β and ‐ε do not.