The expression plasmids for GLK and GLK kinase-dead mutant (GLK-K45E) were reported previously (22). CFP-tagged PKCθ, yellow fluorescent protein (YFP)–tagged AhR, Myc-tagged PKCθ, HA-tagged AhR, and Flag-tagged PKCθ plasmids were constructed by subcloning individual complementary DNAs into pCMV6-AC-CFP, pCMV6-AC-YFP, pCMV6-AC-Myc, pCMV6-AC-HA, or pCMV6-AC-Flag vector (OriGene Technologies). HA-tagged AhR-S36A mutant and Myc-tagged PKCθ-K409W (kinase-dead) mutant plasmids were generated by site-directed mutagenesis. GST-tagged PKCθ and GST-tagged PKCθ-K409W plasmids were also individually constructed by subcloning into pGEX-4T vector. The human GLK shRNA (5′-GTGCCACTTAGAATGTTTGAAA-3′) was obtained from the National RNAi Core Facility (Taiwan) and subcloned into pSUPER-GFP vector (Oligoengine). The IL-17A reporter plasmid was a gift from W. Strober (Addgene plasmid #20124) (28). The IL-17A promoter constructs containing a mutated binding element for AhR, RORγt (−877), RORγt (−120), or STAT3 were generated by site-directed mutagenesis using Fusion DNA polymerase (Thermo Fisher Scientific) according to the manufacturer’s protocol. The following primers were used for mutagenesis (mutated binding elements are underlined and italicized; mutated nucleotides are shown in bold font): AhR-binding site (18), 5′-ATGTCCATACATACATGATACTGAATCACAGC-3′; RORγt-binding site (−887) (28), 5′-CTCAAAGACATAAAGGCAACCGTGATCTCATGGAGAGGAGAG-3′; RORγt-binding site (−120), 5′-GGTTCTGTGCTGCAATCATTTGAGG-3′ (11); and STAT3-binding site (56), 5′-AGACAGATGTTGCCTGTCATAAAGGGGTGGTT-3′.

The mutant constructs were verified by DNA sequencing. The plasmids pGL4.43-Luc2P-XRE (AhR-response XRE-Luc), pGL4.47-Luc2P-SIE (STAT3-responsive SIE-Luc), pGL4.32–Luc2P–NF-κB–RE–Hygro, and pGL4 luciferase reporter vector were purchased from Promega. The plasmid for the RORγt (−877) response element–driven reporter was constructed by cloning four copies of the RORγt (−877) response element into the pGL4.43 luciferase reporter vector. For in vitro binding assays, purified AhR proteins were isolated from HA-AhR–transfected HEK293T cells, followed by HA-peptide elution. Purified recombinant GST-PKCθ and GST-IKKβ proteins were purchased from SignalChem. Purified recombinant 6×His-RORγ proteins were purchased from MyBioSource. Recombinant proteins of GST-tagged PKCθ K409W proteins were isolated from Escherichia coli (BL21) and then purified by GST pulldown assays. Purified Flag-tagged RORγt protein was immunoprecipitated and then eluted with Flag peptides from lysates of HEK293T cells that were cotransfected with Flag-RORγt plus either CFP-IKKβ or vector. Purified recombinant protein of GST-tagged AhR was purchased from Abnova.

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