Disulfide formation in the CaMKII regulatory domain induces autonomy in vitro only if both Cys273 and Cys290 are present in the protein.A, CaMKII mutants' sequences and (B) purified CaMKII Cys273-Cys290 mutant was incubated or not with the thiopyridylated-CaMKII peptide (CaMKII-peptide) and probed with anti-CaMKII antibody (upper panel) or anti-oxidized CaMKII antibody (lower panel). Representative image of n = 3 replicates. C, purified CaMKII Cys273, Cys290, and No Cys mutants were incubated or not with the thiopyridylated-CaMKII peptide and probed with anti-CaMKII antibody (upper panel) or anti-oxidized CaMKII antibody (lower panel). Representative image of n = 3 replicates. D, purified CaMKII Cys273-Cys290, Cys273, Cys290, and No Cys mutants derivatized or not with the thiopyridylated-CaMKII peptide autonomous activity (after EGTA addition). E, purified CaMKII Cys273-Cys290, Cys273, and Cys290 mutants incubated or not with diamide or histamine chloramine in the presence or not of the reducing agents DTT or thioredoxin autonomous activity (after EGTA addition). n = 3 replicates. % of total activity (No EGTA addition). CaMKII, calcium/calmodulin-dependent protein kinase II.