Fig. 4

PAX1 reduces TCF7L2 protein stability. A HEK293FT cells were transfected with increasing amount of PAX1-HA (0, 250, 500, 1000, 1500 ng) expression plasmid and endogenous TCF7L2 levels were analyzed by western blotting. Both bands shown in TCF7L2 blot indicate different splicing isoforms of human TCF7L2 expressed in HEK293FT cells. GAPDH was used as loading control. B Same as (A) except cells were treated with 10 μM CHIR99021 (+CHIR) for 24 hours before harvested. C HEK293FT cells were transfected with TCF7L2-HA and empty vector (−) or PAX1-flag plasmid (+) and 2 days later were treated with 100 μg/ml cycloheximide (CHX) for indicated time and harvested. The expression of TCF7L2 (HA), PAX1 (flag), and GAPDH were analyzed by western blotting. D HEK293FT cells were transfected with empty vectors (−) or PAX1-flag plasmids (+) and cells were treated with 100 μg/ml CHX for indicated time before harvested. Endogenous TCF7L2 levels were analyzed by western blotting. GAPDH was used as loading control. E Same as (D) except cells were treated with 10 μM CHIR99021 (CHIR) for 24 hours before harvested. F Western blotting to analyze the protein levels of indicated proteins in WT and PAX1 overexpression (OE) HCT116 cell lines. GAPDH was used as loading control. Both bands detected in the flag blot should be PAX1 isoforms (NCBI database: NP_006183.2 and NP_001244025.1) which contains 534 and 457 amino acids respectively