Figure 3. Functional analysis of calpastatin phosphorylation using site-directed mutagenesis.

(A) Stable cell lines expressing calpastatin^WT, calpastatin^S655A (non-phosphorylatable mutant), or calpastatin^S655E (phospho-mimetic mutant) were generated in LN18 GBM cells. (B) Western blot of calpain-1 levels in each of the calpastatin stable cell lines and parental LN18 cells 30 seconds and 4 hours after a 10 Gy radiation treatment. (C) Quantitation of calpain-1 levels from two independent western blots (shown in (B) and Supplementary Figure 6A) normalized to total calpastatin. CAST = calpastatin. Untr. = Untreated.