Figure 2. Fusion with a CN peptide enhanced the previously unrecognized direct CaM binding to ant/penetratin. A, Binding of biotinylated CaM (25 nm in TBS, pH 7.5, 1 mm CaCl2) to proteins (CaMKII) and peptides (CBD, CN27, tat, and ant) immobilized by slot blot, in three different dilutions of the amount indicated. B, Interference of peptides (5 μm) with CaM binding (conditions as in A) to brain proteins in a blot overlay assay. The major Ca2+-dependent CaM-binding proteins were CaN-A and CaMKIIα. AntCN27 (top) and ant (bottom) affect Ca2+-dependent CaM binding, but not binding to a protein also detected after Ca2+ was chelated by EGTA. C, antCN27 and ant, but not tatCN21, compete with CBD for binding of TA-labeled CaM. Fluorescence of TA-CaM is reduced after addition of CBD (but not ant; supplemental Fig. 2, available at www.jneurosci.org as supplemental material). In presence of antCN27 or ant (but not tatCN21), significantly more CBD has to be added for the same reduction in fluorescence. Fluorescence (λex = 335 nm; λem = 415 nm; 1 s sample time) was monitored for 150 s after each addition of CBD.