Fig. 3

STAT6 is selectively degraded by Calpain-1 but its PARylation protects it against this process. A Recombinant poly-His-STAT6 was phosphorylated in vitro by active JAK3 or left unphosphorylated prior to incubation with calpain-1 for 10 min. The reaction was stopped with sample buffer and analyzed by immunoblot analysis. The bands were quantified and expressed as percent change compared with their respective untreated control (bottom panel). B Calpain-1 was incubated with STAT6, JAK3, JAK1, PARP-1, or PARG for 40 min. Immunoblot analyses were performed with antibodies against STAT6, PARP-1, JAK1, JAK3, or PARG. C Calpastatin was added to the cell free reaction mix containing STAT6 and Calpain-1. Samples were then subjected to immunoblot analysis with antibodies to the respective proteins. D JAK3-mediated STAT6 phosphorylation, PARylation, and calpain enzymatic reactions were performed in this order. Immediately after the phosphorylation the reaction was divided in 4 parts.Then, immunoblot analysis was carried out with antibodies against STAT6, p-STAT6 (Y641), PARP-1, or PAR. E Recombinant STAT6 was incubated with JAK3 in the presence of calpain-1 with or without NAD⁺. STAT6 degradation was assessed by immunoblot analysis