Journal: Toxicological Sciences

Article Title: MiR-17 Downregulation by High Glucose Stabilizes Thioredoxin-Interacting Protein and Removes Thioredoxin Inhibition on ASK1 Leading to Apoptosis

doi: 10.1093/toxsci/kfv313

Figure Lengend Snippet: Txnip sequesters Thioredoxin from ASK1 under high glucose conditions. A. ASK1 protein abundance in Trx immunoprecipitates from cells treated with different doses of glucose (5 or 25 mM) in the absence or presence of the miR-17 mimic (A), or Txnip siRNA (B) for 48 h. C. ASK1 protein abundance in Trx immunoprecipitates from cells transfected with the miR-17 inhibitor in the absence or presence of Txnip siRNA for 48 h. D. ASK1 and Txnip protein abundances in cells transfected with the Txnip plasmid under normal glucose (5 mM) conditions for 48 h. IP was performed using the anti-Trx antibody. Quantification of IB is shown in the bar graphs. Experiments were repeated 3 times (n = 3). Values are means ± SE from 3 separate experiments. *indicates significant difference (P < .05) compared with the other group(s).

Article Snippet: Membranes were incubated in 5% nonfat milk for 1 h and then were incubated for 18 h at 4°C with the following primary antibodies at dilutions of 1:500–1:5000: Txnip (Cell Signaling Technology, 1:1000), p-ASK1 (Santa Cruz, 1:500), ASK1 (Santa Cruz, 1:1000), Trx (Abcam, 1:1000), and caspase 3 (Millipore, 1:1000).

Techniques: Quantitative Proteomics, Transfection, Plasmid Preparation

Journal: Toxicological Sciences

Article Title: MiR-17 Downregulation by High Glucose Stabilizes Thioredoxin-Interacting Protein and Removes Thioredoxin Inhibition on ASK1 Leading to Apoptosis

doi: 10.1093/toxsci/kfv313

Figure Lengend Snippet: High glucose activates ASK1 through the miR-17-Txnip circuit. A. P-ASK1 protein abundance in cells treated with different doses of glucose (5 or 25 mM) in the absence or presence of the miR-17 mimic for 48 h. IP was performed using the anti-ASK1 antibody. Changes in p-ASK1 were determined by IB. Levels of Txnip mRNA (B) and protein (C) in cells transfected with Txnip siRNA for 48 h. P-ASK1 protein abundance in cells treated with different doses of glucose (5 or 25 mM) in the absence or presence of Txnip siRNA (D), or with co-transfection of the miR-17 inhibitor (E). F. P-ASK1 protein abundance in cells transfected with the Txnip plasmid under normal glucose (5 mM) conditions for 48 h. The quantification of immunoblots is shown in the bar graphs. NC, negative control oligos. Experiments were repeated 3 times (n = 3). Values are means ± SEM from 3 separate experiments. *indicates significant difference (P < .05) compared with the NC group or the other group(s).

Article Snippet: Membranes were incubated in 5% nonfat milk for 1 h and then were incubated for 18 h at 4°C with the following primary antibodies at dilutions of 1:500–1:5000: Txnip (Cell Signaling Technology, 1:1000), p-ASK1 (Santa Cruz, 1:500), ASK1 (Santa Cruz, 1:1000), Trx (Abcam, 1:1000), and caspase 3 (Millipore, 1:1000).

Techniques: Quantitative Proteomics, Transfection, Cotransfection, Plasmid Preparation, Western Blot, Negative Control