Figure 3.

The CAMK4 c.981+1G>A (p.Lys303Serfs*28) allele is expressed in proband-derived cells and engenders a gain-of-function effect. (A) Sanger chromatograms of fibroblast CAMK4 cDNA from proband A-4 and a healthy control (CTRL). Proband A-4's cDNA shows wild-type and c.981+1G>A variant sequence. The nucleotide position at which the 77-bp deletion begins in proband A-4 is indicated with a star. (B) Western blot analysis using an anti-human CaMKIV antibody raised against the amino terminus of the protein. A truncated protein of ∼37 kDa (p.Lys303Serfs*28) was detected in proband A-4's cells (A-4) but not in control cells (CTRL). Boxes represent cropped sections from a digital image of a single membrane (an uncropped version of the image can be found in Supplemental Fig. S1). Two biological replicates are shown for each cell line (CTRL, A-4). (C) Western blot analysis of phospho-CREB and total CREB expression in fibroblasts from proband A-4 (A-4) and a healthy control (CTRL). Ponceau S staining was used as a loading control. Three biological replicates are shown for each cell line (CTRL, A-4). (D) (Bottom) Western blot analysis assessing phospho-CREB and total CREB expression in fibroblasts derived from proband A-4 and a healthy control (CTRL). Cells were treated with vehicle (veh) or STO-609, an inhibitor of CaMKK activity. Boxes represent cropped sections from digital images of the same membrane (uncropped versions of the images can be found in Supplemental Fig. S2). Three biological replicates are shown for each condition (CTRL veh, A-4 veh, A-4 STO-609). (Top) Bar plot showing results of densitometric quantification, depicting the ratio of phosphorylated CREB to total CREB. Data in the bar plot are presented as mean ± standard error of the mean, n = 3.