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. 2015 May 27;27(1):189–201. doi: 10.1681/ASN.2014121241

Figure 4.

Figure 4.

TF regulation by IS and AHR antagonists. (A) IS induces and AHR antagonist suppresses TF within 2 hours of treatment. vSMCs treated with 25 μg/ml IS and 10 μM CH223191 for the indicated time. A representative of three independent experiments is shown. (B) Proteasome inhibition abrogates the effect of an AHR antagonist on both basal and IS-induced TF levels. vSMCs treated for 4 hours with 10 μM proteasome inhibitor (MG132) and 2 hours with 25 μg/ml IS and 10 μM AHR antagonist (CH223191) were lysed and probed. A representative of three independent experiments is shown. (C) AHR antagonist destabilizes IS-induced TF. vSMCs treated with 25 μg/ml IS and CB7993113 for 24 hours were treated with 20 μM emetine and harvested at the indicated time. A representative from three experiments is shown. (D) Densitometry was performed using ImageJ. GAPDH was used to normalize the TF signal. An average of three independent experiments is shown. Error bars=SD. P=0.01 for TF levels compared with and without CB7993113 for a given time point. (E) Suppression of AHR activity enhances TF ubiquitination. vSMCs treated with control or 25 μg/ml IS with or without CB7993113 for 24 hours and MG132 for 4 hours were lysed and probed using TF antibody. A representative of two independent experiments is shown. (F) AHR antagonist increases ubiquitination of basal TF and also restores IS-inhibited TF ubiquitination. vSMCs were treated as above and immunoprecipitated using TF antibody and immunoblotted with ubiquitin antibodies. Immunoprecipitated TF is shown as input. A representative from three experiments is shown. (G) AHR antagonist increases TF ubiquitination. MDA-MB231 cells were transfected with HA-ubiquitin and then treated with 25 μg/ml IS and CB7993113 for 24 hours and 10 μM MG132 for 16 hours. Cells lysed in RIPA buffer were immunoprecipitated using HA antibody and immunoblotted with TF antibody. Five percent of the cell lysates was probed for TF and HA as inputs. A representative immunoblot from three experiments is shown. GAPDH, glyceraldehyde-3-phosphoate dehydrogenase.