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

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© 2016 Lear et al.

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Figure 3. — PIAS4 phosphorylation by PKCζ is required for FIEL1 binding. (A) Endogenous PIAS4 was immunoprecipitated and immunoblotted for Erk1, PKCα, and PKCζ (n = 2). (B) MLE cells were transfected with increasing amounts of PKCζ or JNK1 plasmids for 18 h before PIAS4 immunoblotting (n = 2). (C) PIAS4 protein half-life determination with CON shRNA or PKCζ shRNA expression (n = 3). (D) PIAS4 protein half-life determination with Empty or PKCζ plasmid overexpression (n = 3). (E and F) MRC5 cells were treated with TGFβ in a time or dose-dependent manner; cells were collected and immunoblotted for FIEL1, PIAS4, PKCζ, and p-PKCζ (Thr410). Endogenous PIAS4 was also immunoprecipitated and followed by PKCζ, PKCα, phosphoserine, and phosphothreonine immunoblotting (n = 2). (G) In vitro PKCζ kinase assay. Recombinant PKCζ (Enzo) was used as the kinase, and V5-tagged PIAS4 was 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 PKCζ (n = 2). (H) Immunoblots showing levels of FIEL1, PKCζ, p-PKCζ (Thr410), and PIAS4 protein in 293T cells transfected with either CON shRNA or PKCζ shRNA, followed by a TGFβ dose treatment. Endogenous PIAS4 was also immunoprecipitated and followed by PKCζ, phosphoserine, and phosphothreonine immunoblotting. (I) 293T cells were transfected with WT, S14A, S18A, or S14/18A PIAS4 before being treated with a dose course of TGFβ. Cells were then collected and assayed for V5-PIAS4 immunoblotting. Overexpressed V5-PIAS4 was also immunoprecipitated using a V5 antibody and followed by phosphoserine immunoblotting (n = 2). (J) Four biotin-labeled PIAS4 peptides were bound to streptavidin and served as the bait for FIEL1 binding. After washing, proteins were eluted and immunoblotted for FIEL1-V5 (n = 2).