HEK cells were transfected with CaST and treated with ±50 µM biotin and ±Ca 2+ (5 mM CaCl 2 and 1 µM ionomycin) for 30 min. Cells were then washed with Dulbecco’s phosphate buffered saline (DPBS), and whole-cell lysates were collected and analyzed using a western blot stained with streptavidin–horseradish peroxidase (SA–HRP) or anti-V5/HRP. ‘N’ indicates the expected size of the CaM-V5-sTb(N) fragment, while ‘C’ indicates the expected size of the CD4-sTb(C)-M13-GFP fragment.
