PHD3(H196A) knockin did not affect the expression of p53.
A, RKO cells were transfected with FLAG-tagged mouse PHD3 or PHD3(H196A). After 24 h, the cells were harvested for immunoblotting. B, structure of WT and mutant allele of Phd3 of mice. C, determination of the mutated Phd3 (left panel) and WT Phd3 (right panel) by PCR. Primers 1 and 2 are used for determining the mutated Phd3. Primers 3 and 4 are used for determining the WT Phd3. The homozygous Phd3(H196A) had a band of 1184 bp, and WT Phd3 had a band of 656 bp. The primers for determining WT and mutated Phd3 were shown in Table 1. D, the genotyping of Phd3(H196A)F/F and Phd3(H196A)IEC-KI mice. The primers for determining Villin-Cre was shown in Table 1. E and F, The proteins extracted from small intestine and colon epithelial cells of Phd3(H196A)F/F and Phd3(H196A)IEC-KI mice were subjected to immunoblotting with ATF4 (E) and p53 (F) antibodies. The p53(DO-7) antibody was used to detect mouse p53.