Fig 2: DYRK1A increased NFATc1 protein expression and its nuclear translocation.A. HEK293 cells were transfected with p-NFATc1mycFLAG and pCMV-DYRK1A or pGFP-V-RS-shDYRK1A. NFATc1 expression was detected by mouse anti-FLAG antibody immunoblotting. DYRK1A was blotted with anti-DYRK1A antibody. B. Endogenous NFATc1 was detected by anti-NFATc1 antibody in HEK293 cells transfected with pCMV-DYRK1A. An activated Jurkat T cells treated with 5uM ionomycin plus 500uM calcium chloride for 12 hours was used as positive control. C. HEK293 cells were co-transfected with pWT-NFATc2 and pCMV-DYRK1A. Anti-HA antibody was used to detect NFATc2. D and E. HEK293 cells were co-transfected with p-NFATc1mycFLAG and pCMV-DYRK1A orpGFP-V-RS-shDYRK1A. Mouse anti-FLAG antibody was used to detect NFATc1 protein in nucleus or cytoplasm.?-actin was used as cytoplasmic control and TBP was used as nuclear protein loading control. F. HEK293 cells were treated with ionomycin. Mouse anti-FLAG antibody was used to detect NFATc1 protein in nucleus or cytoplasm ß-actin was used as cytoplasmic control and TBP was used as nuclear protein loading control. Values represent means±SD, n = 3. DY1A means DYRK1A. shDY1A means shDYRK1A.

Fig 3: RCAN1 inhibited NF-?B activity. (a) RCAN1 reduced the luciferase activity controlled by NF-?B. RCAN1.1 and RCAN1.4 expression constructs and RCAN1 siRNA were co-transfected with pNF-?BLuc into HEK293 cells. Renilla luciferase activity was used to normalize transfection efficiency. Dual luciferase assay was performed 48 h after transfection. Values represent mean±S.E.M.; n=4; *P<0.01 by Student's t-test. (b) Total p65 level was increased by RCAN. Plasmids pRCAN1.1/1.4-6myc were co-transfected with pNF-?B into HEK293 cells. Anti-p65 antibody was used to detect the protein level of p65. Anti-myc antibody was used to detect the RCAN1 protein. ß-actin detected by anti-ß-actin was used as loading controls. (c) Quantification of b. Values represent mean±S.E.M.; n=3; *P<0.0001 by Student's t-test. (d, f and g) RCAN1 inhibits NF-?B nuclear translocation. Nuclear proteins were extracted from HEK293 cells co-transfected with expression vectors of NF-?B and pcDNA3.1RCAN1.1/1.4-mychis or si-RCAN1. NF-?B/p65 protein levels were detected by anti-p65 antibody. RCAN1 protein was detected by anti-myc antibody. ß-actin was used as loading controls for cytosol fractions. TBP detected by anti-TBP was used as loading controls for nuclear proteins. (e and h) Quantification of d and g. Values represent mean±S.E.M.; n=3; *P<0.0001 by Student's t-test. (i) The knockdown effect of si-RCAN1. Plasmid si-con or si-RCAN1 was co-transfected with pRCAN1.1-6myc into HEK293 cells, and anti-myc antibody was used to detect the protein level of RCAN1. ß-actin detected by anti-ß-actin was used as loading controls. (j) Quantification of i. Values represent mean±S.E.M.; n=3; *P<0.0001 by Student's t-test. (k) EMSA was performed to analyze the DNA-binding activity of NF-?B. A 20-bp double-strand IRDye 800-labeled consensus NF-?B oligo was used as a probe. Nuclear extracts were derived from HEK293 cells transfected with empty vector, pSi-RCAN1, pRCAN1.1mychis or pRCAN1.4-mychis. (l) Quantification of h. Values represent mean±S.E.M.; n=3; *P<0.001 by Student's t-test

Fig 4: RCAN1 suppressed viability of glioma cells through inhibiting NF-?B signaling pathwayT98G cells were transfected with pSuper-based shRNA plasmid (siRCAN1) or negative control (siCon) for 48 hrs. A. The knockdown effect of psiRCAN1 was verified in protein level by Western blot. RCAN1 was detected with anti-RCAN1 antibody. ß-actin was used as the internal control. The values represented the means ±S.E. (n=3).*p <0.05 by Student's t test. B. Quantification of (A). Values represent the means ± S.E. (n = 3), *p <0.05 by Student's t test. C. Nuclear proteins were extracted. NF-?B/p65 protein levels were detected by anti-p65 antibody. ß-actin was used as loading controls for cytosolfractions. TBP detected by anti-TBP was used as loading controls for nuclear proteins. D. Quantification of (C). Values represent the means ± S.E. (n = 3),*p <0.05 by Student's t test. E. RCAN1 knockdown increased the luciferase activity controlled by NF-?B. These cells were cotransfected with siRCAN1 and pNF-?BLuc. Renilla luciferase activity was used to normalize transfection efficiency. Dual luciferase assay was performed 24 h aftertransfection. Data were calculated from 3 experiments. Values represent the means ± S.E. (n = 3),*p <0.05 by Student's t test. F. Cell viability was measured by MTT assay. The values represent the means ± S.E. (n = 3), p<0.05 by two-way ANOVA.

Fig 5: An Infection-Inducible MEF2-TBP Complex Binds AMP TATA Boxes(A) Requirement for MEF2 and TBP sites in AMP regulation measured by eGFP fluorescence in transgenic flies carrying indicated reporters 8 hr after E. coli injection (general genotype: w1118; ; AMP-eGFP[AttP2]/+). Two male flies are shown for each condition. In site maps, matches to the MEF2 consensus are capitalized, and matches to the TATA consensus are underlined.(B) MEF2 and TBP coimmunoprecipitate from whole flies 3 hr after E cloacae infection; this association is eliminated in Tak1 mutants, but not in Dif; Rel double mutants.(C) MEF2 and TBP bind the Mtk TATA box. The top indicates EMSA with 40 bp oligonucleotides corresponding sequences surrounding the wild-type or non-MEF2-binding mutant (ΔM) TATA box. Nuclear extracts are all from cells expressing MEF2-HA. All lanes are from the same gel and used the same extracts. The bottom indicates a similar experiment, showing that anti-HA and anti-TBP antibodies can inhibit formation of the same complex.(D) TATA box sequences from (top) unbiased 890-TATA sample; (bottom) 27 AMPs and AMP-like factors.(E) Correlation of flanking residues in the 890-TATA sample. Asterisks indicate significant enrichment relative to unconstrained sample (∗p < 0.01; ∗∗p < 0.001; assayed by binomial test).See also Figure S5 and Tables S4 and S5.