CaMKIIα is pulled down more efficiently than Ano1 by CaM-GST. (A–C) Lysates from HEK cells overexpressing CaMKIIα (A), mAno1(ac) (B), or hAno1(ac) (C) were incubated with CaM-GST–Sepharose beads. Lysates were prepared in buffer containing 100 µM Ca²⁺ or EGTA as indicated. Proteins that bound to the beads were detected by immunoblot (IB) with antibodies to hAno1, Flag (for mAno1), or CaMKIIα. Pull-down lanes (two right-most lanes in A–C) contain 1/8 of the protein eluted from the beads. The lysate lanes (left) contain different amounts of lysate expressed as a percentage of the amount of pull-down eluate loaded (for example, 100% represents 1/8 of total lysate). (D) Blot of CaM eluted from beads under same conditions as A–C to show large excess of CaM. Molecular mass is indicated in kilodaltons. (E–G) Quantification of data in A–C. Immunoblots were analyzed by densitometry, and the intensity of each band is expressed as area in relative units. Black lines plot the intensities of different amounts of lysate. Red asterisks indicate the intensity of the pull-down bands in the presence (Ca²⁺) and absence of Ca²⁺ (EGTA). (E) CaMKIIα. (F) mAno1(ac). (G) hAno1(ac).