Fig. 3: IRAK-M–induced apoptosis in melanoma is mediated through calpastatin and calpain.

a Western blot analysis of protein levels of calpastatin, calpain 1 and calpain 2 in melanoma cells transfected with a control or IRAK-M plasmid for 24 h. Changes in calpastatin protein levels in transfected cells are shown. Blots shown are representative of three independent experiments. b Calpain activity in melanoma cells is shown as relative fluorescent units/mg total protein using a fluorescence-based calpain activity assay 24 h after transfection (n = 3 per group). Data are presented as mean ± SEM. **p < 0.01 by two-tailed Student’s t-test. Results are representative of two independent experiments. c Expression levels of apoptosis-related proteins in melanoma cells 24 h after transfection with IRAK-M and/or CAST plasmids by Western blot. Blots are representative of at least two independent experiments. d The calpain activity assay was used to measure calpain activity in melanoma cells transfected for 24 h (n = 3 per group). Results are presented as mean ± SEM. **p < 0.01 by two-tailed Student’s t-test. Data are representative of two independent experiments. e Apoptosis of melanoma cells was determined by flow cytometry (PI and Annexin V staining) 24 h after transfection (n = 3 per group). Results are shown as mean ± SEM. *p < 0.05 and **p < 0.01 by two-tailed Student’s t-test. Data are representative of at least two independent experiments. f–h The effects of 20 µM calpeptin (a calpain inhibitor) treatment in IRAK-M transfected C32 cells for 24 h were evaluated using f western blot, g calpain activity assay (n = 3 per group), and h apoptosis analysis (n = 3 per group). Results are presented as mean ± SEM. **p < 0.01 by two-tailed Student’s t-test. Data presented are representative of at least two independent experiments.