Figure 3
Assessing the system functioning. A-B. HeLa cells expressing βAR and transfected with βarr2-AEQ or with control probes cyt-AEQ and SNAP-AEQ were perfused with a Ca2+-free buffer (KRB/EGTA), showing light emission values slightly above the background (< 100 cps). In control conditions (A) upon Ca2+ re-addition to the medium (KRB/Ca2+) the light emission values changed markedly only for SNAP-AEQ probe, as expected; while upon isoprenaline stimulation (B) also the βarr2-AEQ probe gave rise to a similar light emission increase, confirming probe translocation (βarr2-AEQ control peak 1,730 ± 356 cps n = 9, βarr2-AEQ + isoprenaline peak 6,951 ± 797 cps n = 9; cyt-AEQ control peak 756 ± 36 cps n = 9, cyt-AEQ + isoprenaline peak 562 ± 37 cps n = 9; SNAP-AEQ control peak 8,040 ± 353 cps n = 9, SNAP-AEQ + isoprenaline peak 7,930 ± 80 cps n = 9). C-D. A similar behaviour was observed in HeLa cells over-expressing the PKC βII-AEQ probe. Only upon PMA stimulation (D) an increase in light emission after Ca2+ re-addition was observed, confirming the efficacy of the assay (PKCβII-AEQ control peak 4,115 ± 1,041 cps n = 12, PKCβII-AEQ + PMA 44,140 ± 7,858 cps n = 15; cyt-AEQ control peak 3,884 ± 785 cps n = 10, cyt-AEQ + PMA peak 3,501 ± 519 cps n = 9; SNAP-AEQ control peak 42,517 ± 5,012 cps n = 12, SNAP-AEQ + PMA peak 44,782 ± 5,749 cps n = 11). All results represent cell populations measurements and are expressed as mean ± standard error (SE). The traces showed correspond to a sample representative of the mean obtained from all experiments. n = number of samples (wells) analyzed from at least ten independent experiments.