Fig. 6: ISX-9 prompted BMAL1 activity through S513/515/516 phosphorylation.
From: ISX-9 potentiates CaMKIIδ-mediated BMAL1 activation to enhance circadian amplitude
a Schematic diagram of E-box containing mCamk2 promoter constructs. b Camk2:Luc bioluminescence assay in N2a cells treated with DMSO or 10 μM ISX-9. c Mass spectrometry identification of potential BMAL1 phosphorylation sites responded to ISX-9 treatment in N2a cells. d Amino acid sequence alignment of BMAL1 among the indicated species, S513, S515, and S516 were marked in red, and S519 in blue. e Dbp:Luc bioluminescence assay with expressing BMAL1 WT or mutants in N2a cells. One-way ANOVA with Bonferroni correction for multiple comparison was performed (ns, not significant, ### p < 0.001), and data were shown as mean ± SD. f In vitro kinase assay of purified CaMKIIδ WT and K43M mutant, using BMAL1 as the substrate. g Dbp:Luc bioluminescence assay with expressing CaMKIIδ WT or K43M mutant. b, f, g Unpaired Student’s t-test was used (ns not significant, *p < 0.05, ***p < 0.001), and data were shown as mean ± SD.
