Western Blot of CaM Expression

Western blot was conducted routinely according to the previous description (34). Briefly, proteins were separated in 10% gel by SDS-PAGE and transferred to PVDF (polyvinylidene difluoride, Millipore) using a Hoefer TE70 Semi-Dry Transfer Unit (Pharmacia, San Francisco, CA). After transfer, the membrane was blocked with 5% non-fat dried milk for 1 h and incubated overnight with the primary antibody against human CaM was obtained from Upstate (1:1,000; Milford, MA) at 4°C. The human CaM antibody has been previously used for CaM measurement in fish pituitary cells (29). After washing blots to remove excessive primary antibody binding, the blots were incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody. Blots were developed by SuperSignal WestPico chemiluminescent substrate (Pierce) using the FluorChem FC2 detection system (Biozym Scientific). The densitometric values of bands were analyzed by ImageJ software (NIH). The results represent prototypical examples of experiments replicated at least three times.