Apoptosis was also measured by caspase activation [47 (link)]. The generic caspase activity assay kit (Fluorometric-Green; ab112130) (Abcam, Cambridge, UK) was used to detect the activity of caspase-1, -3, -4, -5, -6, -7, -8, and -9 as described [42 (link)]. Briefly, cells were seeded as 3 × 105 cells per well in 6-well plates with a 2 mL medium for overnight. The cells were then treated with vehicle or TFB. Subsequently, cells were incubated at 37 °C, 5% CO2 for 2 h with 2 μL of 500X TF2-VAD-FMK. After PBS washing, cells were resuspended in 0.5 mL of an assay buffer for immediate flow cytometry measurement (BD Accuri™ C6; Becton-Dickinson).
The apoptosis signaling expressions were further measured via Western blotting. 30 μg protein lysates were resolved in 10% SDS-PAGE. After electrotransferring, the nonspecific bindings of PDVF membranes (Pall Corporation, Port Washington, NY, USA) were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 and incubated with primary antibodies (the cleaved caspase-8 (Asp391) (18C8) rabbit mAb and the rabbit mAb in the apoptosis antibody sampler kit (cleaved PARP (Asp214) (D64E10) XP®; cleaved caspase-3 (Asp175) (5A1E); cleaved caspase-9 (Asp330) (D2D4) from Cell Signalling Technology, Inc., Danvers, MA, USA, β-actin (#GTX629630, GeneTex Inc.)) under 1:10000 dilution as well as their matched secondary antibody. The WesternBright™ ECL HRP substrate (#K-12045-D50, Advansta, Menlo Park, CA, USA) was chosen for signal amplification.
The apoptosis signaling expressions were further measured via Western blotting. 30 μg protein lysates were resolved in 10% SDS-PAGE. After electrotransferring, the nonspecific bindings of PDVF membranes (Pall Corporation, Port Washington, NY, USA) were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 and incubated with primary antibodies (the cleaved caspase-8 (Asp391) (18C8) rabbit mAb and the rabbit mAb in the apoptosis antibody sampler kit (cleaved PARP (Asp214) (D64E10) XP®; cleaved caspase-3 (Asp175) (5A1E); cleaved caspase-9 (Asp330) (D2D4) from Cell Signalling Technology, Inc., Danvers, MA, USA, β-actin (#GTX629630, GeneTex Inc.)) under 1:10000 dilution as well as their matched secondary antibody. The WesternBright™ ECL HRP substrate (#K-12045-D50, Advansta, Menlo Park, CA, USA) was chosen for signal amplification.
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