Axioobserver inverted confocal microscope

Fixed neurons were permeabilized by incubation with cold methanol at −20°C for 5 min and blocked with 2% BSA in PBS at RT for 1 hr. Neurons were immunostained against mouse-anti-V5 (1:2000 dilution, Invitrogen) and rabbit-anti-VP16 (1:2000 dilution, Abcam), followed by anti-mouse-alexafluoro488 (1:1000 dilution) and anti-rabbit-alexafluoro647 (1:1000 dilution) in 2% BSA solution in PBS. Neurons directly plated on the 48-well plate were imaged with 10x air objective in the Zeiss AxioObserver inverted confocal microscope and neurons plated on glass cover slips were imaged with 40x oil objective in the Zeiss AxioObserver inverted confocal microscope. Eight to ten fields of view were collected for each condition.

HEK293T cells were cultured on coverslips pretreated with human fibronectin. For the evolved LOV conditions, HEK293T cells were transfected with a mix of DNA constructs P16 (50–100 ng/well, Table SI-1), P7 (15 ng/well, Table SI-1), P9 (15 ng/well, Table SI-1) in 10 μL DMEM and 0.8 μL PEI max. For the original LOV, HEK293T cells were transfected with a mix of DNA constructs P11 (50–100 ng/well, Table SI-1), P7 (15 ng/well, Table SI-1), P9 (15 ng/well, Table SI-1) in 10 μL DMEM and 0.8 μL PEI max. HEK293T cells were stimulated 18 hrs post transfection under four conditions as above, light+high calcium, light+low calcium, dark+high calcum, dark+low calcium. HEK293T cells were fixed and immunostained as above. HEK293T cells were then imaged on an imaging dish with 40x oil objective in the Zeiss AxioObserver inverted confocal microscope.