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. 2022 Apr 27;298(6):101986. doi: 10.1016/j.jbc.2022.101986

Figure 4.

Figure 4

CK1γ3 kinase activity is required for maximum WNT3A activation.A, HEK293T B/R cells were transfected with the indicated construct for 14 h, treated with Lcell or WNT3A CM for 18 h, and then analyzed by luciferase assay. B, live-cell imaging of HEK293T cells stably expressing a BAR-GFP fluorescent reporter transiently transfected with the indicated expression construct, CK1γ3-WT or CK1γ3-K72A. Lcell or WNT3A CM was added at 8 h, and cells were monitored for an additional 72 h. Data are averaged across four replicates. C, HEK293T cells transfected with either CK1γ3-WT or CK1γ3-K72A for 14 h then either untreated or treated with WNT3A CM for 30 or 60 min. Samples were then analyzed by W. blot. D and E, quantification of W. blot from A for indicated time points, pLRP6 T1479 (D) and pLRP6 S1490 (E) across four biological replicates, normalized to total LRP6. All statistics are compared to FLAG-CK1γ3 Lcell sample. For all panels: ∗∗∗p < 0.0005, ∗∗p < 0.005, and ∗p < 0.05. All biological replicates are represented in box-and-whisker plots, with the boxes representing the median and the interquartile range. All biological replicates are averaged across three technical replicates. Statistical significance was evaluated by Student’s t test. CK1γ3, casein kinase 1γ3; CM, conditioned media; HEK293T, human embryonic kidney 293T cell line; LRP6, low-density lipoprotein receptor–related protein 6; W, blot, Western blot.