GSK3β regulates PIAS4 protein stability through FIEL1. (A) Endogenous FIEL1 was immunoprecipitated and followed by JNK2, PKCα, and GSK3β immunoblotting (n = 2). (B) MLE cells were transfected with increasing amounts of WT or constitutively activated GSK3β hyper mutant plasmids for 18 h before PIAS4 immunoblotting. The arrow indicates the overexpressed GSK3β (n = 2). (C) PIAS4 protein half-life determination with WT GSK3β or hyperactive GSK3β plasmid overexpression. The arrow indicates the overexpressed GSK3β (n = 2). (D) MRC5 cells were treated with TGFβ in a time or dose-dependent manner; cells were then collected and immunoblotted for FIEL1 and PIAS4. Endogenous FIEL1 was also immunoprecipitated and followed by phosphoserine and phosphothreonine immunoblotting (n = 2). (E) PIAS4 protein half-life determination with CON shRNA or GSK3β shRNA expression (n = 2). (F) Immunoblots showing levels of GSK3β, phospho-GSK3β (Ser9), PIAS4, and FIEL1 protein in 293T cells transfected with either CON shRNA or GSK3β shRNA followed by a TGFβ dose treatment. Endogenous FIEL1 was also immunoprecipitated, followed by phosphoserine and phosphothreonine immunoblotting. (G) Lung samples from Fig. 1 J were subjected to FIEL1 immunoprecipitation, followed by PIAS4, phosphothreonine, and phosphoserine immunoblotting. PIAS4 protein abundance was plotted as a function of p-Thr protein (n = 5 patients per group; *, P < 0.01, Pearson correlation).