Figure 2

(a) Effect of Rad, CsA, FK506, VER and HA9 on the ADP-ribosylation status of Giα in PT-treated CHO-K1 cells. Cells were pre-incubated with 20 µM of Rad, CsA or FK506, 30 µM VER, 100 µM HA9 or 20 µM BFA for control for 30 min or left untreated. Then, cells were challenged with 20 ng/ml PT for 3 h. Cells were lysed and ADP-ribosylation status of Giα was analyzed. Biotin-labeled (i.e., ADP-ribosylated) Giα was detected. Comparable protein loading was confirmed by Hsp90 and Ponceau S staining. Western blot signals were quantified and normalized to protein loading (Ponceau S). One representative result is shown (n = 3). For uncropped blots see Supplemental Fig. S10. (b) Effect of chaperone inhibitors on intracellular cAMP levels in PT-treated cells. iGIST sensor cells (HEK293 cells expressing SSTR2 and luminescent cAMP probe) were incubated with CsA or FK506 (5 µM) or left untreated for control for 30 min. 500 ng/ml PT were added for 3 h and afterwards inducing medium was added to start the luminescence reaction. A baseline was recorded for 15 min. Then, cells were treated with forskolin to activate adenylate cyclase and with octreotide to activate SSTR2. Luminescence was recorded for 1 h. Values are given as mean ± SD. Results from one representative experiment are shown. (c) Effect of Rad, CsA, FK506 and VER on enzyme activity in vitro. Recombinant Giα (0.5 µM) was pre-incubated with 10 µM Rad, CsA, 20 µM FK506 or VER for 30 min or with buffer for control. After 30 min rPTxS1 (50 nM) and 1 µM biotin-labeled NAD⁺ were added and incubated for 40 min at room temperature. Samples were subjected to SDS-PAGE, blotted and ADP-ribosylated Giα was detected with Strep-POD. Western blot signals were quantified. Values are given as percent of samples treated with PTS1 only (n = 3 values from 3 independent experiments, mean ± SD). Significance was tested one-way ANOVA with Dunnett’s multiple comparisons test and refers to samples treated with PTS1 only (ns not significant). For uncropped blot see Supplemental Fig. S11. (d) Influence of Rad, CsA, FK506 and VER on receptor binding of PT to CHO-K1 cells. CHO-K1 cells were pre-incubated with 20 µM Rad, CsA, FK506 or 30 µM VER or left untreated. After 30 min cells were cooled to 4 °C for 15 min. Then 500 ng/ml PT were added for 30 min at 4 °C. After washing, cells were lysed with Laemmli buffer at 95 °C. Cell-bound PT was detected via an anti-PTS1 antibody in Western Blot. Comparable protein loading was confirmed with Hsp90 and Ponceau-S-staining. Quantification of Western blot signals and one representative Western blot is shown. Values were normalized on the amount of loaded protein (Ponceau S) and are given as percent of PT binding (second bar from left). (n = 4, mean ± SEM). Significance was tested by one-way ANOVA with Dunnett’s multiple comparisons test and refers to samples treated with PT only (ns not significant). For uncropped blots see Supplemental Fig. S12.