Fig. 1

Quantitative scanning of MarsCy1-tagged membrane proteins in a modular plate-based format. a Top: Cartoon depicting HA-FAP fused to the N-terminus of the CD80 transmembrane domain. Bottom: Confocal imaging of FAP-CD80 transfected cells stained with SCi1. b Cartoon depicting HA-FAP fused to the N-terminus of Lgr5 and EGFP fused to the C-terminus. c Confocal imaging of FAP-tagged Lgr5-EGFP transfected cells that were co-labeled with SCi1 (magenta) and a primary HA-epitope antibody (Red, secondary retrieval – 568 nm) at 4 °C to block receptor internalization and were chased in (d) for 30 minutes at 37 °C to allow constitutive internalization of Lgr5 (EGFP, green) (K44A, A dominant-negative Dynamin I mutant that inhibits receptor endocytosis when over-expressed). e Infrared plate imaging of a 12-well plate (see Additional file 4: Figure S4 for entire plate and time-course) with FAP-tagged Lgr5 pulsed with SCi1 and a primary HA-epitope antibody at 4 °C and then fixed. Non-permeabilized cells were scanned on the plate at 700 nm (red, SCi1) and 800 nm (green, HA secondary retrieval-800 nm) (NT: non-transfected). f Integrated fluorescence intensity from (e). g MarsCy1-tagged hV2R was transiently transfected in HEK cells on a 24-well plate and stimulated with vehicle (–AVP) or the V2R ligand AVP [10 μM] for 1 hour. Cells were SCi1 stained, scanned, and quantified