Figure 6

The effect of Bip, Calreticulin and ERp57 on G-protein assembly with the heterodimer. HEK293 cells were transfectedwith β2AR-v1, AT1R-v2 and Rluc G-proteins (Gαs, Gαi, Gβ1 and Gγ2). 48 hours post transfection, cells were harvested, washed three times with PBS then suspended in 100 μl PBS. 90 μl of the samples was distributed into 96 well plates and 10 μl of 5 μM coelenterazine H was added and mixed then the fluorescence energy emitted by Rluc and YFP (venus) was measured at their corresponding emission wavelengths (460 and 528 nm, respectively). The BRET ratio was then calculated and plotted with the ratios for a negative control (β2AR-v1, AT1R-v2 and the pRluc-N3 vector) and a positive control (β2AR-Rluc and β2AR-EGFP). Results are expressed as means ± sem of at least 3 experiments. Statistical analysis was performed using two-tailed paired Student's t test.