NFATc1 mutants abolished the effects of DYRK1A.

<p><b>A.</b> Nine mutant constructs of NFATc1 were co-transfected with pCMV-DYRK1A in HEK293 cells. <b>B.</b> HEK293 cells co-transfected with NFATc1 and nine mutant constructs of NFATc1 were treated with proteasome inhibitor lactacystin (5uM) for 24 hours after transfection. Values represent means±SD, n = 3. DY1A means DYRK1A. ubi means ubiqutin. pSer means phosphorylated serine.</p

DYRK1A affected NFATc2 protein phosphorylation.

<p><b>A.</b> DYRK1A inhibitor harmine (2ug/ml) decreased NFATc1 expression (the image was flipped horizontally from the original picture). <b>B.</b> Anti-pSer antibody was used to detect the serine phosphorylated NFATc1 and rabbit anti-FLAG antibody was used to detect the total NFATc1 in the mouse anti-FLAG antibody immunoprecipitates. Mouse anti-DYRK1A antibody and mouse anti-NFATc1 antibody were used to detect total DYRK1A and total NFATc1, respectively in transfected cells. <b>C.</b> Co-IP assay showed the cross-interaction between NFATc1 and DYRK1A. <b>D.</b> Anti-ubiquitin antibody was used to detect the ubiquitination of NFATc1 and rabbit anti-FLAG antibody was used to detect the total NFATc1 in mouse anti-FLAG antibody immunoprecipitates. Mouse anti-DYRK1A antibody and mouse anti-NFATc1 antibody were used to detect total DYRK1A and total NFATc1, respectively in transfected cells, Values represent means±SD, n = 3. Ubi means ubiqutin. pSer means phosphorylation of serine.</p

Identification of DYRK1A-targeted motifs in NFATc1 protein.

<p><b>A.</b> Diagram showed NFATs sequence alignment. Sequences of NFATc1 (NP_765978), NFATc2 (NP_775114), NFATc3 (NP_775118), and NFATc4 (NP_001129494) were aligned using ClustalW2 software. Conserved regions among NFATs are underlined. Conserved serine residues in four NFATs are shown in blue. The DYRK1A target motifs are labeled as yellow highlights. The DYRK1A phosphorylation serine residues were in red and bold. <b>B.</b> Schematic diagram represents the domain structure of NFATc1 and location of five deletion constructs. AD: activation domain. SPRIEIT: calcineurin binding motif. SP: sp repeats region; SRR: serine-rich region; NLS: nuclear localization sequence. <b>C.</b> Five deletion constructs of NFATc1 were co-transfected with pCMV-DYRK1A in HEK293 cells. <b>D.</b> Deletionconstructs of NFATc1 was co-transfected with pCMV-DYRK1A in HEK293 cells. Anti-DYRK1A antibody was used as the immunoprecipitation antibody, and anti-FLAG antibody was used to detect NFATc1 in immunoblotting.</p

DYRK1A affected NFATc2 protein ubiquitination and stability.

<p><b>A and B</b>. HEK293 cells transfected with p-NFATc1mycFLAG were treated with proteasome inhibitorlactacystin (lac) for time or dosage indicated. <b>C and D</b>. HEK293 cells transfected with p-NFATc1mycFLAG were treated with lysosome inhibitor chloroquine (chl) for time or dosage indicated. <b>E.</b> Co-IP assay showed the cross-interaction between NFATc1 and ubiquitin. <b>F and G.</b> NFATc1 and NFATc2 were increased by inhibition of SCF E3 ligase using Cullin-1/2 DN constructs. Values represent means±SD, n = 3.Lacmeanslactacystin. Ubi means ubiqutin.</p