To test the split enzyme’s reversibility, HEK cells were transfected with CaST-IRES and treated with biotin alone for 30 min (top), with Ca 2+ for 30 min followed by a 10-min wash and then biotin for 30 min (middle), or with biotin + Ca 2+ simultaneously for 30 min (bottom). Example images are shown for all three conditions with SA-647 staining of biotin and GFP expression of CaST-IRES. Scale bar, 300 µm.
