Hibit assay kit

Cells (13,000 per well) were plated on 96-well plates (Greiner Bio-one) coated with polyornithine (10 μg/mL). After 10 h, the cells were co-transfected with a plasmid expressing Shh-SBP-HiBiT (or a secreted control, SecGFP-SBP-HiBiT) under doxycycline control and a plasmid constitutively expressing the hook (Strepta, core streptavidin-KDEL provided with a signal peptide). Some of these cells received an additional plasmid constitutively expressing a membrane-bound fusion between mCherry and DAO (Lck-Che-DAO). After 24 h, the medium was removed and cells were incubated with fresh medium containing doxycycline for 2 h (to induce HiBiT fusion expression). Medium was changed and secretion was induced with biotin (100 μM final) and, after the purified large fragment of split nanoluciferase (LgBiT) protein addition to the medium, the luciferase activity was measured 1 h later with a 96-well plate luminometer (Tristar, Berthold) as described in the HiBiT assay kit (Promega). The cells were then lysed to measure the intracellular protein expression. Normalization with biotin-untreated wells enabled us to calculate the secretion index and report the secretion efficiency.

Cells (90,000 per well) stably expressing LgBiT were plated in 24-well culture dishes. After 24 h, the medium was changed and cells were incubated for 30 min with the medium containing HiBiT fusions (taken from cells expressing Shh-HiBiT or secreted mCherry, SecCh-HiBiT for 48 h, and adjusted on protein Luciferase activity), before incubating the cells with trypsin and removing them. After centrifugation, cells were lysed and the luciferase activity of endocytosed protein was measured with a 96-well plate luminometer (Tristar, Berthold) with a HiBiT assay kit (Promega).

Cells (90,000 per well) were plated in 24-well culture dishes. After 10 h, the cells were co-transfected with a plasmid expressing Shh-SBP-HiBiT under the control of doxycycline and a plasmid constitutively expressing Lck-Che-DAO. After 24 h, cells were removed using trypsin and co-cultured on 96-well plates (Greiner Bio-one) coated with polyornithine (10 μg/mL) with cells stably expressing LgBiT anchored to the extracellular side of the cell surface (siL-LgBiT-mb5). After 5 h, cells were incubated with fresh medium containing doxycycline for 2 h (to induce Shh-SBP-HiBiT expression). Medium was then changed and secretion was induced with biotin (100 μM final), and luciferase activity was measured every 1 h over 4 h with a 96-well plate luminometer (Tristar, Berthold) as described in the HiBit assay kit (Promega).