Impact of Cell Type and Epitope Tagging on Heterologous Expression of G Protein-Coupled Receptor: A Systematic Study on Angiotensin Type II Receptor
Figure 5
Ubiquitination of transiently expressed epitope-tagged AT2 receptors.
(A, B) The HA-ubiquitin expression construct (5 µg) were transiently co-transfected with Myc-AT2 expression construct or control empty vector (5 µg) into HEK293 cells (in a 100-mm dish); or HA-ubiquitin expression construct (5 µg) was transiently transfected into HEK293 cells (in 100-mm dish) that stably expressed Myc-AT2 or control HEK293 cells that stably transfected with empty vector. After 48 hr, cells were incubated with 10 µg/ml MG132 for 5 hr and then lysed in 1 ml of RIPA buffer containing 20 mM N-ethylmaleimide. Cell lysate was subject to immunoprecipitation with an anti-Myc antibody and then probed with an (A) anti-HA or (B) anti-Myc antibody. (C, D) Expression constructs of HA-ubiquitin (5 µg) and AT2-GFP (5 µg) were transiently co-transfected into HEK293 cells (in 100 mm dish); or HA-ubiquitin expression construct (5 µg) was transiently transfected into HEK293 cells that stably expressed AT2-GFP (in a 100-mm dish). After 48 hr, cells were lysed in 1 ml of RIPA buffer. Cell lysate was subject to immunoprecipitation with an anti-GFP antibody and then probed with an (C) anti-HA or (D) anti-ubiquitin antibody. Ab VH: Antibody heavy chain. Data shown is representative of 2–3 independent experiments with similar results.