Fig. 2. STUB1 destabilizes and ubiquitinates TF and mediates TF regulation by AHR. STUB1 destabilizes and ubiquitinates TF and mediates TF regulation by AHR. (A) Lysates from primary human aortic vSMCs transfected with control (Csi) or STUB1 silencing oligonucleotides (STUB1si) were probed for TF and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using ImageJ. Equal amounts of lysates were separately probed for STUB1 to avoid stripping of blots for the proteins with molecular weights in close range, a strategy also applied to other figures. Expression of TF and STUB1 normalized to the loading control is shown below this and subsequent Western blots. A representative figure from four independent experiments is shown. (B) vSMCs pretransfected with Csi and STUB1si were stimulated with 5% of the indicated type of serum for 24 hours. A mean TF activity of two independent experiments performed in duplicates is shown. Student’s t test was performed. Number sign (#) corresponds to TF activity in Csi vSMCs treated with uremic compared to control serum. Other P values correspond to STUB1-silenced compared to Csi cells. Data are shown as means ± SD. (C) vSMCs pretransfected with Csi and STUB1si grown in 5% uremic serum for 24 hours and then treated with cycloheximide (80 μM) to inhibit protein translation for the indicated time. Equal amounts of lysates were probed separately to confirm STUB1 silencing. TF expression normalized to the loading control is depicted below the blot. A representative figure from four independent experiments is shown. (D) Densitometry of normalized TF expression is represented as the percentage of TF at time 0. The time to reach 50% of initial TF was considered as the half-life of TF. An average of four experiments is shown. Data are shown as means ± SD. (E) vSMCs pretransfected with Csi and STUB1si were treated with 5% uremic serum with or without the AHR antagonist CB7993113 (20 μM) or vehicle for 24 hours. A representative of two independent experiments performed in duplicate is shown. (F) vSMCs pretransfected with Csi and STUB1si were treated with 5% uremic serum + 20 μM CB7993113 (12 hours) and 10 μM MG132 (4 hours) before harvest. The lysates immunoprecipitated with anti-TF antibody were probed with anti–ubiquitin (Ub) antibody. Five percent of lysates are shown as inputs. A representative of three independent experiments is shown. IP, immunoprecipitation; WB, Western blotting. Moshe Shashar et al., Sci Transl Med 2017;9:eaam8475 Published by AAAS