Journal: bioRxiv

Article Title: Cryo-EM structure of cell-free synthesized human histamine H 2 receptor coupled to heterotrimeric G s protein in lipid nanodisc environment

doi: 10.1101/2023.07.27.550782

Figure Lengend Snippet: a. Schematic of GPCR nanotransfer. b. H 1 R-mCherry synthesized after transfection internalizes upon histamine treatment (100 µM histamine for 1h; + H) and co-localizes with nanotransferred and CF synthesized H 2 R-mNG (right panel). White bar = 10 µm. c. Representative images of HEK293T cells transferred for 4 h with 0.5 µM purified H 2 R-mNG in NDs (DOPG) and treated with histamine. Left panel: Localization of transferred H 2 R-mNG in untreated cells. Right panel: Internalization of transferred H 2 R after incubation with 100 µM histamine for 1h. White bar = 10 µm. d. Quantification of cytosolic fluorescence of transferred H 2 R-mNG. Data are presented as mean s.d. (n = 3 individual experiments, *p < 0.05, students t-test). e. Interaction of transferred CF synthesized GPCRs with transfected H 1 R and H 2 R. Transfected cells expressing Flag-tagged H 1 R or H 2 R were transferred with CF synthesized Strep-tagged H 1 R, H 2 R or FFAR 2 for 4 h. Subsequently, cells were washed, lysed and transferred GPCRs were immobilized by anti-Strep pulldowns. Input: Immunoblots of cell lysates before anti-Strep pulldowns. Pulldown: Immunoblots of immobilized GPCRs after anti-Strep pulldowns. Strep: Anti-Strep antibody; Flag: Anti-Flag antibody; Control: Cells without transfection (a. created by biorender).

Article Snippet: Next, cells were incubated with a rabbit anti-FLAG M2 antibody (142 ng/mL) (Cell Signaling Technology, Danvers, MA, USA) in 1% BSA–PBS for 1 h at 4 °C.

Techniques: Synthesized, Transfection, Purification, Incubation, Fluorescence, Expressing, Western Blot, Control