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. 2016 May 30;213(6):1029–1046. doi: 10.1084/jem.20151229

Figure 5.

Figure 5.

GSK3β phosphorylation of FIEL1 is required for PIAS4 targeting. (A) MLE cells were transfected with increasing amounts of WT, T783A, or T783D mutant FIEL1 plasmids for 18 h before PIAS4 immunoblotting (n = 2). (B) PIAS4 protein half-life determination with WT, T783A, or T783D mutant FIEL1 (n = 2). (C) In vitro GSK3β kinase assay. Recombinant GSK3β (Enzo) was used as the kinase, and V5-tagged FIEL1 were synthesized via TnT in vitro kits (Promega), purified by HIS pulldown, and used as the substrate. The kinase reactions were incubated at 37°C for 2 h, and products were resolved by SDS-PAGE and processed for autoradiography either by using Personal Molecular Imager (Bio-Rad Laboratories) or immunoblotting for V5 to visualize the substrate input. *heat inactivated GSK3β (n = 2). (D) 293T cells were transfected with empty, WT, or T783A FIEL1 for 24h. Cells were then collected and immunoblotted for V5-FIEL1 and PIAS4. Overexpressed V5-FIEL1 was also immunoprecipitated using V5 antibody and followed by phosphothreonine immunoblotting (n = 2). (E) Four biotin-labeled FIEL1 peptides were prebound to streptavidin and served as the bait for PIAS4 binding. After washing, proteins were eluted and processed for PIAS4 immunoblotting (n = 2). (F) MLE cells were transfected with increasing amounts of WT, T783A, P779L, or T783A/P779L double mutant FIEL1 plasmids for 18 h before PIAS4 immunoblotting. (G) PIAS4 protein half-life determination with WT, T783A, P779L, or T783A/P779L double mutant FIEL1 (n = 2). (H) PIAS4 peptide 2 (Biotin-MSFRVS(p)DLQM) was prebound to streptavidin and served as the bait for FIEL1 binding. After washing, proteins were eluted and processed for V5-FIEL1 immunoblotting (n = 2). (I) FIEL1 peptide 2 (Biotin-QIIAAPTHST(p)LPTA) was bound to streptavidin and served as the bait for PIAS4 binding. After washing, proteins were eluted and processed for V5-PIAS4 immunoblotting (n = 2). (J) 293T cells were transfected with WT, T783A, P779L, or T783A/P779L double mutant FIEL1 before being treated with TGFβ. Cells were then collected and assayed for PIAS4 immunoblotting. (K) MLE cells were transfected with increasing amounts of WT, I514V, or D207V mutant FIEL1 plasmids for 18 h before PIAS4 immunoblotting. (L) PIAS4 protein half-life determination with WT, I514V, or D207V mutant FIEL1 expression.