Journal: Cell Death Discovery
Article Title: ER stress-induced cell death proceeds independently of the TRAIL-R2 signaling axis in pancreatic β cells
doi: 10.1038/s41420-022-00830-y
Figure Lengend Snippet: A Cell-death measurements in L929, Min6 and Ins1E cells treated with hexavalent TRAIL in presence of sensitizer cycloheximide (CHX, 1 µg/ml). PI uptake served as a read-out for cell death. Data show mean ± SEM from n = 3 independent experiments. B Amplification cycles from qPCR studies including independent Tnfrsf10b primer pairs. Displayed are Ct-values of the respective qPCRs on RNA without (−) or with (+) 1 µM TG stimulation for 24 h prior to RNA isolation. Red background indicates increasingly unspecific amplifications. C Immunoblot comparison of TRAIL-R2 protein amounts between L929, Min6 and Ins1E cells without (−) or with (+) 1 µM TG stimulation for 24 h prior to protein isolation. D Min6 cells expressing doxycycline-inducible human TRAIL-R2 or control cells were stimulated with increasing concentrations of doxycycline. TRAIL-R2 expression was monitored by immunoblotting. EV, empty vector. E Cell-death measurements after treatment of Min6 cells expressing human TRAIL-R2 with hexavalent TRAIL, 1 µg/ml cycloheximide (CHX) and 50 µM QVD. PI uptake served as a read-out for cell death. Cells were pre-treated with 0.1 µg/ml doxycycline for 24 h. Shown is one representative graph, mean ± SD of two independent experiments.
Article Snippet: The following primary antibodies were used for western blotting: Rat monoclonal BIP (1:500, 76-E6, BioLegend Inc., San Diego, California, USA), mouse monoclonal CHOP/GADD 153 (1:200, B-3, Santa Cruz Biotechnology Inc., Dallas, Texas, U.S.A.), mouse monoclonal Vinculin (1:500, 7F9, Santa Cruz Biotechnology Inc., Dallas, Texas, U.S.A.), rabbit polyclonal TRAIL-R2 (1:500, ab8416, Abcam, Cambridge, UK), rabbit monoclonal TRAIL-R2 (1:800, D4E9, #8074), mouse monoclonal alpha-Tubulin (1:10 000, DM1A, # 3873), rabbit monoclonal MCL-1 (1:1 000, D2W9E, # 94296), rabbit polyclonal Caspase-3 (1:1000, # 9662), rabbit polyclonal PARP (1:1 000, # 9542) all from Cell Signaling (Danvers, MA, USA), mouse monoclonal cleaved PARP (1:700, Asp214, #51–9000017, BD Biosciences, Heidelberg, Germany).
Techniques: Amplification, Isolation, Western Blot, Comparison, Expressing, Control, Plasmid Preparation
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