Dynamin

Mouse brains were homogenized in the modified RIPA buffer containing protease inhibitors (Thermo Scientific). Equal amounts of proteins from wild-type and CaN_Aα^-/- mice, determined by BCA protein assay, were loaded onto SDS-PAGE gel and immunoblotted using antibodies against CaN_Aα (1:200, Santa Cruz Biotechnology), clathrin (1:1,000, BD Bioscience), dynamin (1:1,000, BD Bioscience), AP2 (1:100, Thermo Scientific), endophilin (1:200, Invitrogen) and actin (1: 400, Abcam).

For Western blot, neurons were washed three times with ice-cold PBS. Cell lysates were prepared in the modified RIPA buffer containing protease inhibitors (Thermo Scientific, Rockford, IL). Equal amounts of proteins, determined by BCA protein assay (Thermo Scientific, Rockford, IL), were loaded onto SDS-PAGE gel and immunoblotted using antibodies against clathrin heavy chain (1:1,000; BD Bioscience, San Jose, CA), dynamin (1:1,000; BD Bioscience, San Jose, CA, recognizing all dynamin isoforms, including 1, 2 and 3), dynamin 1 (1:1,000; Thermofisher, Waltham, MA) and β-actin (1:2,000; Abcam, Cambridge, United Kingdom).