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. 2020 Jan 20;27(7):2131–2142. doi: 10.1038/s41418-020-0490-7

Fig. 2. PHD3 stabilized ATOH1.

Fig. 2

a Determination of NICD, HES1, and ATOH1 in intestinal epithelial cells by western blot. The small intestinal epithelial and colonic epithelial cells were isolated as described in “Methods”. b Determination of the mRNA levels of Hes1 in intestinal epithelial cells (n = 6). c CCD841 cells were transfected with myc-PHD3 vector. After 24 h, the cells were harvested for determination of ATOH1 by western blot. d The cells were transfected with PHD3 siRNA oligos as indicated. After 48 h, the cells were harvested for determination of ATOH1 by western blot. e CCD841 cells were transfected with myc-PHD3 vector. After 24 h, the cells were harvested for determining ATOH1 expression by qPCP. f CCD841 cells were transfected with PHD3 siRNA oligos. After 48 h, the cells were harvested for determining ATOH1 expression by qPCR. g Determination of mRNA levels of Atoh1 in mice intestinal epithelial cells (n = 6). h 293T cells were transfected with Flag-ATOH1 or Flag-ATOH1 plus myc-PHD3 vector. After 24 h, the cells were harvested for western blot. i 293T cells were transfected with Flag-ATOH1 or Flag-ATOH1 plus myc-PHD3 plasmid. After 24 h, cycloheximide (CHX) (10 μg/ml) was added and the cells were incubated for different time interval as indicated. j The relative Flag-ATOH1 level was determined by measuring the density of Flag-ATOH1 band and normalized to that of actin. The relative Flag-ATOH1 protein level at the starting time is designated as 1.