Fig. 4: IRAK-M-induced apoptosis in human melanoma depends on TRAF6-mediated recruitment of calpastatin.

a Expression levels of indicated proteins were detected by Western blot in melanoma cells 24 h after transfection with empty vector, WT His-IRAK-M or His-IRAK-M-ΔCTD (a truncated His-IRAK-M variant lacking the entire CTD domain). Blots are representative of three independent experiments, each yielding identical trends. b 24 h after transfection, calpain activity (relative fluorescent units/mg total protein) in melanoma cells was evaluated using the fluorescence-based calpain activity assay (n = 3 per group). Data are shown as mean ± SEM. **p < 0.01 by two-tailed Student’s t-test. Results are representative of two independent experiments. c Apoptosis of melanoma cells transfected for 24 h was measured by staining cells with PI and Annexin V, followed by flow cytometry (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 shown are representative of at least two independent experiments. d–f The effects of TRAF6 knockdown (shTRAF6) in C32 cells 24 h after transfection were determined by d western blot, e calpain activity assay (n = 3 per group), and f 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. g–i Melanoma cells were transfected with the indicated vectors for 18 h and collected for IP assay and western blot. Cell lysates were subjected to IP assay using sepharose bead conjugated His-tag mouse antibody and western blot using the indicated antibodies. Images shown here are representative of at least two independent experiments. j Western blot was performed to detect the expression levels of TRAF6 and calpastatin proteins in IRAK-M transfected C32 cells treated with 20 µM of the proteasome inhibitor (R)-MG132 for 24 h. Blots are representative of at least two independent experiments.