Detailed steps of western blotting were previously described [60 (link)]. Briefly, the primary apoptosis antibodies (diluted 1:1000) including cleaved caspase-3 (c-Cas 3) rabbit mAb (Cell Signaling Technology, Inc., Danvers, MA, USA) were used. The internal control primary antibody (diluted 1:5000) was mAb-β-actin (Sigma-Aldrich, St. Louis, MO, USA). Following secondary antibody treatment, these protein signals were detected using enhanced chemiluminescence (ECL) substrate (WesternBright™ ECL HRP, Advansta, Menlo Park, CA, USA).
For c-Cas 3-based flow cytometry, cells were fixed with 70% ethanol, washed, and incubated with 1 μg/mL of c-Cas 3 (Asp175) rabbit mAb (Cell Signaling Technology) at 4 °C for overnight. After washing, cells were incubated with a secondary polyclonal antibody conjugated with Alexa Fluor 488 (ThermoFisher Scientific, San Jose, CA, USA) at room temperature for 1 h. Finally, the c-Cas 3 expression was analyzed by Accuri™ C6 flow cytometry. Cas 8 inhibitor Z-IETD-FMK (100 μM, 2 h) or Cas 9 inhibitor Z-LEHD-FMK (100 μM, 2 h) were applied to examine the involvement of Cas 8 and Cas 9 in apoptosis.
For c-Cas 3-based flow cytometry, cells were fixed with 70% ethanol, washed, and incubated with 1 μg/mL of c-Cas 3 (Asp175) rabbit mAb (Cell Signaling Technology) at 4 °C for overnight. After washing, cells were incubated with a secondary polyclonal antibody conjugated with Alexa Fluor 488 (ThermoFisher Scientific, San Jose, CA, USA) at room temperature for 1 h. Finally, the c-Cas 3 expression was analyzed by Accuri™ C6 flow cytometry. Cas 8 inhibitor Z-IETD-FMK (100 μM, 2 h) or Cas 9 inhibitor Z-LEHD-FMK (100 μM, 2 h) were applied to examine the involvement of Cas 8 and Cas 9 in apoptosis.
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